New Generation Vaccines, Fourth Edition

New Generation Vaccines

New Generation Vaccines Fourth Edition Editor-in-Chief

Myron M. Levine

Center for Vaccine Development, University of Maryland School of Medicine Baltimore, Maryland, USA Editors

Gordon Dougan

The Wellcome Trust Sanger Institute Hinxton, Cambridge, UK

Michael F. Good

Queensland Institute of Medical Research Queensland, Australia

Margaret A. Liu

ProTherImmune and Karolinska Institutet Lafayette, California, USA

Gary J. Nabel

Vaccine Research Center, National Institutes of Health Bethesda, Maryland, USA

James P. Nataro

Center for Vaccine Development, University of Maryland School of Medicine Baltimore, Maryland, USA

Rino Rappuoli

Novartis Vaccines and Diagnostics Siena, Italy

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2010 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-4200-6073-2 (Hardcover) International Standard Book Number-13: 978-1-4200-6073-7 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data New generation vaccines / edited by Myron M. Levine ; associate editors, Gordon Dougan . . . [et al.]. — 4th ed. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-1-4200-6073-7 (hb : alk. paper) ISBN-10: 1-4200-6073-2 (hb : alk. paper) 1. Vaccines. I. Levine, Myron M. (Myron Max), 1944[DNLM: 1. Vaccines. QW 805 N5316 2009] QR189.N489 2009 6150 372—dc22 2009034901 For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

Foreword

As this eagerly awaited fourth edition of New Generation Vaccines goes to press, the world is facing the greatest global financial crisis in three-quarters of a century. This prompts deep thinking about societal priorities and human goals. The ‘‘greed is good’’ syndrome has largely disappeared and the rampant individualism dominant in industrialized societies for 50 years is being questioned. It is no longer politically incorrect to state that multinational corporations should have twin goals: profit for the shareholders and good citizenship within their spheres of influence. In this context, it is easier to get a debate going about inequities in health. Most will agree that the two to three decade gap between the introduction of a new generation vaccine in the rich countries and its availability in the third world is simply no longer acceptable. Given that vaccines represent history’s most cost-effective public health tools, shortening that gap becomes an urgent priority. Given further that many vaccines can be made available to developing countries only through aid flows, public and private, it is imperative that the global financial crisis not be allowed to impede the momentum that has gathered around this issue over the last 10 to 15 years. A world that can afford trillion dollar bailouts of banks and other companies can easily afford the several billion per year required to immunize all the world’s children. Prioritization, of course, cannot be avoided. This fact makes the updated New Generation Vaccines particularly welcome and valuable. Lent a unique authority by its distinguished editor and associate editors, its 89 chapters cover every aspect of the vast field of vaccinology. While the science underpinning vaccination in general and individual vaccines in particular occupies the bulk of the space, the vitally important social issues involved in global immunization are also dealt with in some detail. Decision-makers charged with establishing priorities are thus helped by up-to-date scientific evaluation of vaccines buttressed by sober analysis of economic, logistic, regulatory, industrial, ethical, and political aspects. In its 130-year history as an independent discipline, immunology has displayed a curious capacity to reinvent itself, perhaps about once a decade. In the most recent period, three themes have dominated: the extraordinary relationship between the ancient, innate immune system and the adaptive system confined to vertebrates; cellular regulation of immune responses with ever better defined subsets of interacting lymphocytes and antigen-presenting cells; and biochemical signaling cascades with 100 or 200 agonists interacting with cellular receptors and initiating sequential reactions culminating in gene activation or silencing. It is reassuring to note that these themes have been richly picked up, where relevant, in their many practical applications to vaccine design and development. The volume begins with a brief historical perspective and then vaults straight to the most modern way of defining putative vaccine antigens, namely ‘‘reverse vaccinology,’’ made possible by the genomic revolution. Then follows a detailed discussion of vaccine clinical trials. As an unashamed advocate for vaccines, I have had a long experience with anti-vaccine activists, particularly in the media, who love controversy. Of course the sharpest weapon in my armory is the exacting process of phased clinical trials, elaborately aimed at establishing risks and benefits. It is good to see this area given even greater prominence in the fourth edition. A series of chapters then deals with important social, economic, industrial, and regulatory matters. It gave me special pleasure to view the impressive progress of the GAVI Alliance, the international cooperative effort that is galvanizing immunization in the 72 poorest countries of the world. I had the privilege of being involved in the planning and early implementation phases of GAVI, and its success is most heartening. The global eradication of smallpox is still one of the major triumphs of public health, so it is fitting that the first chapter devoted to a given disease is bringing us up-to-date with polio eradication. There could not be a more powerful illustration of

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the interface between science and sociology. The idealism and extraordinary volunteer effort behind polio eradication surely deserves to be crowned with success. An extensive series of chapters explores the interface between fundamental immunology and vaccinology including the importance of certain platform technologies, the influence of age, and the problem of autoimmunity. This leads naturally to a deep consideration of antigen delivery systems—formal adjuvants on the one hand, physicochemical methods on the other, as well as a cornucopia of viral and bacterial vector systems. Furthermore, a future where more of our vaccines can be given mucosally or transdermally is explored; this is important. We risk becoming the victims of our own success. As more vaccines prove themselves, infants receive more injections and mothers become troubled. The issue of combinations and of schedules is skillfully explored. Appropriately, as the book moves to a consideration of specific diseases and vaccines, several chapters deal first with carbohydrate-protein conjugate vaccines. Beginning with Haemophilus influenzae type b, these have represented real breakthrough products, worth reporting in considerable detail. One of the world’s biggest challenges is how to deploy 10- to 13-valent pneumococcal conjugate vaccines in the third world as pneumonia still kills 1.8 million people annually. Fortunately, a novel funding mechanism known as Advanced Marketing Commitments is addressing this problem with an initial $1.5 billion. Then the book deals with many novel viral and bacterial vaccines bringing us right up-to-date with progress and not shying away from the immense challenges that still lie ahead for ‘‘difficult’’ diseases like HIV/AIDS or tuberculosis. The field of parasitology comes next, with heartening progress in malaria reported and exciting work in leishmaniasis, schistosomiasis, amebiasis, and hookworm. As a reflection on our troubled world, there is an extensive section on vaccines against agents of bioterrorism and an adventurous exploration of the field of emerging infectious agents. Applied immunology simply will not allow itself to be confined to communicable diseases. Vaccines against cancer are no longer science fiction. ‘‘Negative’’ vaccines to prevent autoimmunity are the subject of extensive clinical trials. Vaccines against neurodegenerative disorders are at an earlier stage of research but remind us how unexpectedly immunological principles can pop up in other organ systems. And who would have thought that immunotherapy might represent an approach to drug addiction? These provocative chapters provide a fitting conclusion to the book. The editors and authors are to be congratulated on having achieved a fine balance between authoritative comprehensiveness and great accessibility. This is because the editors have an Olympian overview of the field and the authors have been chosen as internationally renowned and committed experts in their specialties. The work is both theoretically satisfying and practically useful. The fourth edition of New Generation Vaccines is thus a rare triumph and a vindication of the immense effort that has gone into it. It is an essential acquisition for any library, institutional or personal, purporting to be serious about supporting vaccine science. Given the importance of vaccines to global health, it will be a precious guide to many seeking to create a better world. G. J. V. Nossal The University of Melbourne, Victoria, Australia

Preface

Vaccinology continues, impressively, to advance and mature both in the development of new and improved vaccines and in the implementation of vaccines to prevent disease, thus requiring the publication of the new fourth edition. Some technologies highlighted with great expectation in the previous edition have progressed admirably in clinical trials, whereas others have proved disappointing and have been abandoned altogether. Some of the important changes that have taken place in the field and are featured in this edition include several new vaccines that have been recently licensed, including a quadrivalent (groups A, C, W135, and Y) meningococcal conjugate vaccine, two oral rotavirus vaccines, and two human papillomavirus (HPV) vaccines based on virus-like particle technology. Certain already existing vaccines, with modification, for example, acellular pertussis, have been adapted from use in infants for use in adolescents and adults. New fundamental knowledge on the intricacies of the innate immune system, in particular the role of Toll-like receptors, has revolutionized understanding of the relationship between the innate and adaptive immune systems, thereby providing a scientific underpinning to elevate adjuvant research from being largely empiric to becoming science based. This suggests that the next few years may see breakthroughs to enhance immune responses to poorly immunogenic vaccine antigens and to increase immunologic responses to vaccines in the very young and the elderly target groups that have, heretofore, been notoriously less immunologically responsive. Technologies that allow high-throughput sequencing and bioinformatic analysis of genome sequence data have advanced at a frenzied pace. Many pathogens of interest for vaccine development have had their genomes sequenced, thereby allowing searches to be undertaken to identify antigens as potential targets to serve as vaccine candidates. The fourth edition very much highlights these technologies. This edition also continues the book’s tradition of providing extensive descriptions of various live bacterial and viral vector vaccine strategies and technologies. The entry of the Bill and Melinda Gates Foundation into the arena of vaccine development and implementation was recorded in the previous edition. In the ensuing years, the Foundation has greatly expanded the scope and breadth of its activities and has become a major supporter of research and development for HIV, malaria, and tuberculosis vaccines, as well as the primary funding source for an array of other vaccine development efforts for neglected diseases, including cholera, Shigella and enterotoxigenic Escherichia coli diarrheal pathogens, dengue, and Japanese encephalitis B virus, group A meningococcus, measles among infants too young to receive the currently licensed vaccine and hookworms. The fourth edition provides an updated report on the extraordinary impact of the Global Alliance for Vaccines and Immunization (now called the GAVI Alliance) and its financial instrument, the Vaccine Fund. GAVI has become an established fact on the ground in developing countries and represents one of the most significant initiatives in vaccine public health since the establishment of the Expanded Program on Immunization in the 1970s. The GAVI Alliance partners, including the World Health Organization (WHO), UNICEF, the World Bank, the Bill and Melinda Gates Foundation, vaccine industry in both industrialized and developing countries, and others, are committed to increasing immunization coverage among infants in developing countries by strengthening the infrastructure of immunization services, introducing new vaccines (e.g., Haemophilus influenzae type b conjugate) into developing country programs and fostering the accelerated development and introduction of vaccines that can diminish young child mortality (multivalent pneumococcal conjugate and rotavirus vaccines). The fourth edition also updates both the impressive progress and the frustrating setbacks of the global Polio Eradication Initiative in recent years.

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At the time of publication of the fourth edition, the supply of safe and effective vaccines was undergoing a fundamental change that reflected the dichotomy in the array of vaccines routinely administered to infants in the developing world versus infants and toddlers in the industrialized countries. Major vaccine producers in the developing world, most of which have evolved from government-supported facilities to become private profit-making entities, have successfully assumed the responsibility of providing most of the routine vaccines needed for infants in developing countries. These include major producers in India, China, Indonesia, and Brazil. That trend has continued and has reached the point where several of the more sophisticated vaccine manufacturers in developing countries have embarked on impressive research and development programs to construct, prepare pilot lot formulations, undertake large-scale manufacture process development, perform clinical trials, and submit to licensure by regulatory agencies several new vaccines targeted for use in developing countries. The fourth edition will relate several examples of such projects, including a monovalent meningococcal A conjugate and a method for delivering small particle–aerosolized attenuated measles vaccine. The increasing role of manufacturers in the developing world in supplying vaccines for populations in those countries has resulted in the need to strengthen national regulatory agencies in those countries where vaccines are manufactured. Accordingly, a chapter has been added to the fourth edition to relate WHO’s efforts to strengthen national regulatory agencies. In addition, the excellent chapter on the FDA as an example of the roles and responsibilities of a regulatory agency in handling biologics has been expanded. The issue of ‘‘vaccine safety’’ represents another area of striking dichotomy between vaccines used in the industrialized countries versus the developing world. In industrialized countries, where infectious diseases such as measles, poliomyelitis, and pertussis are rare, consequent to decades of immunization coverage, some segments of the population are becoming more focused on rare real or perceived adverse events related to vaccines rather than being concerned about the disease the vaccine aims to prevent. The trend is to expect ever higher levels of safety in relation to the use of vaccines. In contrast, in developing countries where measles and pertussis remain killers and polio stills paralyzes some unvaccinated individuals in certain countries, the emphasis is on protection against these diseases. As more countries transition to become industrialized, views about vaccine safety will undoubtedly change. These issues are becoming more relevant as it is increasingly recognized that the world is a ‘‘global village’’ where modern travel allows pathogens that are still prevalent in developing countries but rare in industrialized countries to reappear with a vengeance in the latter if immunization coverage falls too low. Several chapters in the fourth edition acknowledge the notion that disease control is a shared global responsibility. The previous edition had multiple chapters describing progress on therapeutic vaccines against chronic immunopathological diseases such as rheumatoid arthritis, multiple sclerosis and type 1 diabetes mellitus and of vaccinotherapy for cancer. In the ensuing years, these efforts have yielded many disappointments along with a few breakthroughs; so there is now considerable scientific retrenchment. The fourth edition of NGV addresses these changes with two scholarly, unbiased overview chapters that review, respectively, the immunotherapy of chronic diseases and cancer vaccinotherapy and relate progress in these areas. The fourth edition ratains several chapters that relate progress on the development of vaccines against certain other chronic progressive pathologies such as Alzheimer’s disease. The last edition also described preclinical studies with candidate antiaddiction vaccines. The fourth edition relates the progress as these vaccines have progressed to clinical trials with encouraging results and also addresses ethical issues that have arisen over the use of such vaccines. One of the most significant changes in vaccine development since the last edition is in the area of vaccines against the highest threat bioterror agents and some emerging infectious disease agents, including human pandemic influenza. Notable investments in vaccine development have been made by the U.S. government to support the development of new vaccines against anthrax, smallpox, tularemia, an array of hemorrhagic fever viruses, and other emerging viruses (e.g., Ebola, Hendra, West Nile, SARS) and plague, as well as against other pathogens of potential bioterror concern (e.g., Shigella dysenteriae 1 and other Shigella). Accompanying these specific vaccine development efforts has been the corollary development of

Preface

improved methods of immunizing populations en masse, in particular without the use of needles and syringes. The fourth edition has made a concerted effort to cover these exciting developments by including multiple chapters devoted to marking their progress. Even with 89 chapters, it is impossible to include in a single text of research vaccinology updates on vaccines against every disease target and all new technologies. Thus, as with previous editions, there are some areas and topics where progress has been made that we have been unable to include. The editors have tried to minimize these voids. The editors would like to thank the many contributors who have provided outstanding chapters for the fourth edition. The editors extend their special thanks to family members, friends, and colleagues whose support and patience sustained our efforts to complete this edition during the many evening and weekend hours required to plan and edit this edition. The book could not have been completed without the competent assistance and diligence of Mrs Dottie Small and the ever available support, advice, and counsel of Ms Sandra Beberman and Aimee Laussen of Informa Healthcare. Myron M. Levine, Gordon Dougan, Michael F. Good, Margaret A. Liu, Gary J. Nabel, James P. Nataro, and Rino Rappuoli

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Foreword G. J. V. Nossal . . . . . . . . . v Preface . . . . . . . . . . . vii Contributors . . . . . . xvii 1. Vaccines and Vaccination in Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Myron M. Levine, Rosanna Lagos, and Jose´ Esparza 2. Developing Vaccines in the Era of Reverse Vaccinology . . . . . . . . . . . . . . . . . . . . . 12 Rino Rappuoli, Gordon Dougan, and Isabel Delany 3. Initial Clinical Evaluation of New Vaccine Candidates . . . . . . . . . . . . . . . . . . . . . . 25 Carol O. Tacket and Karen L. Kotloff 4. Special Issues in Performing Vaccine Trials in Developing Countries . . . . . 34 Milagritos D. Tapia, Karen L. Kotloff, Christopher V. Plowe, and Samba O. Sow 5. Long-Term Evaluation of Vaccine Performance: Methodological Issues for Phase III and Phase IV Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 John D. Clemens, Abdollah Naficy, Malla R. Rao, and Hye-won Koo 6. Ethical Considerations in the Conduct of Vaccine Trials in Developing Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Charles Weijer, Claudio F. Lanata, and Christopher V. Plowe 7. Vaccine Economics: Assuring That Vaccines Are Developed for and Available in Developing Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Amie Batson and Piers Whitehead 8. Development and Supply of Vaccines: An Industry Perspective . . . . . . . . . . . . 79 Michel Gre´co and Luc Hessel 9. Reaching Every Child: Achieving Equity in Global Immunization . . . . . . . . . 91 R. Bruce Aylward, B. Melgaard, M. Birmingham, J. Lloyd, R. Eggers, J. Bilous, and T. Cherian 10. A Paradigm for International Cooperation: The GAVI Alliance . . . . . . . . . . . . 101 Lisa Jacobs 11. Economic Analyses of Vaccines and Vaccination Programs. . . . . . . . . . . . . . . . . 107 Benjamin Schwartz and Mark L. Messonier 12. An Overview of U.S. Food and Drug Administration Licensure of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Lydia A. Falk, Norman Baylor, and Karen Midthun 13. Assuring Vaccine Quality by Strengthening Regulatory Agencies: The Work of the World Health Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Julie Milstien, Nora Dellepiane, Lahouari Belgharbi, Liliana Chocarro, and David Wood 14. Vaccine Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Neal A. Halsey, Robert Ball, and Leslie K. Ball

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15. Manufacturing of Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Guido Dietrich, Julie Milstien, Suresh Jadhav, and Peter C. Stowe 16. Polio Eradication: Ongoing Innovation to End an Ancient Scourge R. Bruce Aylward, Stephen L. Cochi, and David L. Heymann

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17. Recent Advances in Immunology That Impact Vaccine Development . . . . . 166 Marcelo B. Sztein, Rafi Ahmed, and Shane Crotty 18. Modulating Vaccine Responses with Innate Immunity . . . . . . . . . . . . . . . . . . . . . 183 Bali Pulendran, Jeff Powell, and Richard A. Flavell 19. Immunodominance, Deceptive Imprinting, and Immune Refocusing Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Peter L. Nara and George Lin 20. Standardization and High-Throughput Measurement of T-Cell Responses to Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 Kent J. Weinhold and Janet S. Ottinger 21. Transition to High-Throughput Laboratory Assays to Evaluate Multivalent Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Sandra Romero-Steiner, George M. Carlone, Helen Baxendale, and David Goldblatt 22. The Challenge of Vaccine Protection in Very Young Infants . . . . . . . . . . . . . . . 227 Claire-Anne Siegrist 23. The Challenge of Inducing Vaccine Protection in the Elderly . . . . . . . . . . . . . . 234 Dietmar Herndler-Brandstetter, Angelika Schwanninger, and Beatrix Grubeck-Loebenstein 24. Vaccination and Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 Paul-Henri Lambert and Michel Goldman 25. Adjuvants for the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 Richard T. Kenney and Alan S. Cross 26. TLR9 Agonists for Immune Enhancement of Vaccines . . . . . . . . . . . . . . . . . . . . . . 263 Heather L. Davis 27. Use of Genetically or Chemically Detoxified Mutants of Cholera and Escherichia coli Heat-Labile Enterotoxins as Mucosal Adjuvants. . . . . . . . . . . 273 Lucy C. Freytag, John D. Clements, Dubravka Grdic Eliasson, and Nils Lycke 28. Recent Developments in Nonliving Antigen Delivery Systems . . . . . . . . . . . . 284 Derek T. O’Hagan 29. Virus-Like Particles as Vaccines and Vaccine Delivery Systems . . . . . . . . . . . 298 Kathrin U. Jansen, Margaret E. Conner, and Mary K. Estes 30. Subunit Vaccines Produced Using Plant Biotechnology Qiang Chen, Hugh S. Mason, Tsafrir Mor, Alec Sutherland, Guy A. Cardineau, Charles J. Arntzen, and Carol O. Tacket

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31. Lipopeptide-Based Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 David C. Jackson, Brendon Y. Chua, Lorena E. Brown, Peter Moyle, and Istvan Toth 32. Vaccines Based on Dendritic Cell Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Silvia B. Boscardin, Michel C. Nussenzweig, Christine Trumpfheller, and Ralph M. Steinman

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33. Vaccinia Virus and Other Poxviruses as Live Vectors . . . . . . . . . . . . . . . . . . . . . . . 340 Bernard Moss 34. Replication-Defective and Competent Adenovirus Recombinants as Vaccine Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 Marjorie Robert-Guroff, Gary J. Nabel, and John W. Shiver 35. RNA Virus Replicon Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 Nancy L. Davis and Robert E. Johnston 36. Engineering of Attenuated Salmonella enterica Serovars for Use as Live Vector Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 James E. Galen, Marcela F. Pasetti, Sharon Tennant, Marcelo B. Sztein, and Myron M. Levine 37. DNA Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 Gary J. Nabel, David C. Kaslow, Jeffrey B. Ulmer, and Margaret A. Liu 38. Overview of Heterologous Prime-Boost Immunization Strategies . . . . . . . . . . 396 Helen McShane, Adrian V. S. Hill, and Andrew McMichael 39. Mucosal Immunization and Needle-Free Injection Devices . . . . . . . . . . . . . . . . . 405 Jakub K. Simon, Myron M. Levine, Bruce G. Weniger, and Ana M. H. Restrepo 40. Advances in Transcutaneous Vaccine Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415 Robert C. Seid, Jr. and Gregory M. Glenn 41. Rationalizing Childhood Immunization Programs: The Variation in Schedules and Use of Combination Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 Andrew J. Pollard, Kathryn M. Edwards, and Bernard Fritzell 42. Meningococcal Conjugate and Protein-Based Vaccines Andrew J. Pollard, E. Richard Moxon, and Rino Rappuoli

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43. Post-Licensure Impact of Haemophilus influenzae Type b and Serogroup C Neisseria meningitidis Conjugate Vaccines in Industrialized Countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452 Elizabeth Miller, Helen Campbell, and Mary Ramsay 44. Haemophilus influenzae Type b Disease Burden and the Impact of Programmatic Infant Immunization in Developing Countries . . . . . . . . . . . . . . 464 Richard A. Adegbola, Samba O. Sow, and J. Anthony G. Scott 45. Pneumococcal Protein-Polysaccharide Conjugate Vaccines Cynthia G. Whitney and Orin S. Levine

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46. Pneumococcal Common Proteins and Other Vaccine Strategies . . . . . . . . . . . . 482 David E. Briles, James C. Paton, Susan K. Hollingshead, and John W. Boslego 47. Polysaccharide-Based Conjugate Vaccines for Enteric Bacterial Infections: Typhoid Fever, Nontyphoidal Salmonellosis, and Escherichia coli O157:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489 Shousun C. Szu, John. B. Robbins, Rachel Schneerson, and F.-Y. Lin 48. Attenuated Strains of Salmonella enterica Serovars Typhi and Paratyphi as Live Oral Vaccines Against Enteric Fever . . . . . . . . . . . . . . . . 497 Myron M. Levine, James E. Galen, Marcela F. Pasetti, and Marcelo B. Sztein 49. Oral Cholera Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506 Jan Holmgren and James B. Kaper 50. Novel Vaccines Against Tuberculosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516 Marcus A. Horwitz, Peter Andersen, and Stefan H. E. Kaufmann

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51. Influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 John Treanor, Wendy Keitel, and Iain Stephenson 52. Chimeric Vaccines Against Japanese Encephalitis, Dengue, and West Nile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557 Konstantin V. Pugachev, Farshad Guirakhoo, Dennis W. Trent, and Thomas P. Monath 53. Vaccines Against Rotavirus Gastroenteritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570 Richard L. Ward, H. Fred Clark, Paul A. Offit, Umesh D. Parashar, and Roger I. Glass 54. Novel Strategies for Immunizing Infants in Developing Countries Who Are Too Young to Receive the Currently Licensed Measles Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Marcela F. Pasetti, Karina Ramirez, Karen L. Kotloff, Eileen M. Barry, and Myron M. Levine 55. Challenges and Prospects for the Development of an HIV Vaccine Barney S. Graham and Gary J. Nabel

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585

56. Vaccine Strategies to Prevent Dengue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593 Robert Edelman, Stephen S. Whitehead, and David W. Vaughn 57. Vaccination Against the Hepatitis C Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Sergio Abrignani, Raffaele De Francesco, and Michael Houghton 58. Vaccines Against Respiratory Syncytial Virus and Parainfluenza Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620 Alexander Schmidt, Emmalene Bartlett, Anne Schaap-Nutt, Peter L. Collins, Brian R. Murphy, and Ruth Karron 59. Cytomegalovirus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633 David I. Bernstein and Stanley A. Plotkin 60. Epstein–Barr Virus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646 Viviana P. Lutzky and Denis J. Moss 61. Herpes Simplex Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653 David M. Koelle, Lawrence R. Stanberry, Reuben S. Carpenter, and Richard J. Whitley 62. Development of Vaccines to Prevent Group A Streptococcal Infections and Rheumatic Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672 J. B. Dale, M. Batzloff, Michael F. Good, J. McCarthy, J. R. Carapetis, P. P. Cleary, V. Fischetti, and J. Zabriski 63. Vaccines Against Group B Streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690 Morven S. Edwards, Carol J. Baker, and Lawrence C. Paoletti 64. Overview of Live and Subcellular Vaccine Strategies Against Shigella . . . . 700 Karen L. Kotloff, Eileen M. Barry, Myron M. Levine, Thomas L. Hale, and Philippe Sansonetti 65. Vaccines Against Enterotoxigenic Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . 714 Ann-Mari Svennerholm and Gregory Glenn 66. Multivalent Shigella Enterotoxigenic Escherichia coli Vaccine . . . . . . . . . . . . . . 723 Eileen M. Barry and Myron M. Levine 67. Vaccines for Staphylococcus aureus Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728 Juliane Bubeck Wardenburg, Dominique Missiakas, and Olaf Schneewind

Contents

68. Chlamydia trachomatis Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Renata Grifantini and Guido Grandi 69. Malaria Vaccines in Clinical Development: Introduction and Recombinant/Subunit Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746 Laura B. Martin, Robert W. Sauerwein, Teun Bousema, David E. Lanar, and Thomas L. Richie 70. Gene-Based Malaria Vaccines Adrian V. S. Hill

.................................................

774

71. Pre-erythrocytic and Asexual Erythrocytic Stage Whole-Organism Malaria Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783 Stephen L. Hoffman, B. Kim Lee Sim, Adam Richman, Alberto Pinzon-Charry, Michael F. Good, and J. McCarthy 72. Vaccines Against Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 790 Farrokh Modabber, Rhea Coler, and Steven G. Reed 73. Vaccines Against Schistosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 808 Alex Loukas, Jeffrey M. Bethony, Andre´ Capron, and Donald P. McManus 74. Vaccines Against Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820 Christopher D. Huston and William A. Petri, Jr. 75. Hookworm Vaccines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830 Jeffrey M. Bethony, Maria Elena Bottazzi, Ami Shah Brown, David Diemert, Peter Hotez, and Alex Loukas 76. Improved Smallpox Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838 Matthew E. Cohen and Stuart N. Isaacs 77. Anthrax Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851 Arthur M. Friedlander, Stephen F. Little, and Marc Gurwith 78. Tularemia Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 864 Alan S. Cross and Eileen M. Barry 79. Vaccines Against Plague . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 871 Scott A. Lloyd 80. Development of Vaccines for Ebola and Marburg Viruses: Efficacy and Regulatory Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 879 Gary J. Nabel 81. Therapeutics and Vaccines Against Hendra and Nipah Viruses Christopher C. Broder

...........

885

82. Vaccines Against Lassa Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 Maria S. Salvato and Igor S. Lukashevich 83. Hantavirus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905 Christopher D. Hammerbeck and Jay W. Hooper 84. SARS Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914 Kanta Subbarao 85. Cancer-Specific Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927 Drew Pardoll 86. Vaccines Against Human Papillomaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954 Kathrin U. Jansen and Ian H. Frazer

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Contents

87. Vaccines Against Alzheimer’s and Other Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 962 Roy M. Robins-Browne and Colin L. Masters 88. Vaccination for Autoimmune and Other Chronic Inflammatory Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969 Leonard C. Harrison 89. Immunotherapies To Treat Drug Addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982 Paul R. Pentel, Dan E. Keyler, and Thomas R. Kosten Index . . . . 993

Contributors

Sergio Abrignani INGM, Istituto Nazionale Genetica Molecolare, Milano, Italy Richard A. Adegbola Bill & Melinda Gates Foundation, Seattle, Washington, U.S.A.; and formally Medical Research Council (U.K.) Laboratories, The Gambia Rafi Ahmed Emory Vaccine Center, Emory University School of Medicine, Emory University, Atlanta, Georgia, U.S.A. Peter Andersen Department of Infectious Diseases Immunology, Staten Serum Institut, Copenhagen, Denmark Charles J. Arntzen Arizona, U.S.A.

The Biodesign Institute, Arizona State University, Tempe,

R. Bruce Aylward Department of Vaccines and Biologicals, World Health Organization, Geneva, Switzerland Carol J. Baker Section of Infectious Diseases, Departments of Pediatrics and of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A. Robert Ball Center for Biologics Evaluation and Research, Office of Biostatistics and Epidemiology, Food and Drug Administration, Rockville, Maryland, U.S.A. Leslie K. Ball U.S.A.

Center for Drug Evaluation and Research, Rockville, Maryland,

Eileen M. Barry Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Emmalene Bartlett Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, U.S.A. Amie Batson Health, Nutrition, Population, Human Development Network, The World Bank, Washington, D.C., U.S.A. M. Batzloff

Queensland Institute of Medical Research, Queensland, Australia

Helen Baxendale

Institute of Child Health, London, U.K.

Norman Baylor Center for Biologics Evaluation and Research, FDA, Rockville, Maryland, U.S.A. Lahouari Belgharbi Department of Immunization, Vaccines and Biologicals, Quality, Standards and Safety Team, World Health Organization, Geneva, Switzerland David I. Bernstein Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, U.S.A. Jeffrey M. Bethony Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A.

xviii

Contributors

J. Bilous Department of Immunizations, Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland M. Birmingham

World Health Organization Country Office, Bangkok, Thailand

Silvia B. Boscardin Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, U.S.A. Vaccine Development Global Program, PATH, Washington, D.C.,

John W. Boslego U.S.A.

Maria Elena Bottazzi Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A. Teun Bousema Department of Medical Microbiology, Radbound University Nijmegen Medical Center, Nijmegen, The Netherlands David E. Briles Departments of Microbiology and Pediatrics, The University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Christopher C. Broder Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland, U.S.A. Ami Shah Brown Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A. Lorena E. Brown Department of Microbiology & Immunology, The University of Melbourne, Parkville, Victoria, Australia Helen Campbell Immunisation Department, Centre for Infections, Health Protection Agency, London, U.K. Acade´mie des Sciences De´le´gation aux Relations Internationales,

Andre´ Capron Paris, France

J. R. Carapetis Menzies School of Health Research, Charles Darwin University, Darwin, Australia Guy A. Cardineau Arizona, U.S.A.

The Biodesign Institute, Arizona State University, Tempe,

George M. Carlone Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Reuben S. Carpenter Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, New York, U.S.A. Qiang Chen U.S.A.

The Biodesign Institute, Arizona State University, Tempe, Arizona,

T. Cherian Department of Immunizations, Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland Liliana Chocarro Department of Immunization, Vaccines and Biologicals, Quality, Standards and Safety Team, World Health Organization, Geneva, Switzerland Brendon Y. Chua Department of Microbiology & Immunology, The University of Melbourne, Parkville, Victoria, Australia H. Fred Clark Department of Pediatrics, Division of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. P. P. Cleary University of Minnesota, Minneapolis, Minnesota, U.S.A.

Contributors

John D. Clemens

International Vaccine Institute, Seoul, Korea

John D. Clements Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana, U.S.A. Stephen L. Cochi Global Immunization Division, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Matthew E. Cohen Department of Medicine, Division of Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Rhea Coler Infectious Disease Research Institute, Seattle, Washington, U.S.A. Peter L. Collins Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, U.S.A. Margaret E. Conner Department of Molecular Virology and Microbiology and Veterans Affairs Medical Center, Houston, Texas, U.S.A. Alan S. Cross Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Shane Crotty La Jolla Institute for Allergy & Immunology, La Jolla, California, U.S.A. J. B. Dale University of Tennessee Health Science Center, Veterans Affairs Medical Center, Memphis, Tennessee, U.S.A. Heather L. Davis Pfizer Vaccines Research Unit, Ottawa, Ontario, Canada Nancy L. Davis Carolina Vaccine Institute, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A. Raffaele De Francesco INGM, Istituto Nazionale Genetica Molecolare, Milano, Italy Isabel Delany Siena, Italy

Molecular Genetics Unit, Novartis Vaccines and Diagnostics S.R.L.,

Nora Dellepiane Department of Immunization, Vaccines and Biologicals, Quality, Standards and Safety Team, World Health Organization, Geneva, Switzerland David Diemert Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A. Guido Dietrich Berna Biotech Ltd., Berne, Switzerland, and GlaxoSmithKline Biologicals, Wavre, Belgium Gordon Dougan Microbial Pathogenesis, Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K. Robert Edelman Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Kathryn M. Edwards Department of Pediatrics, Division of Pediatric Infectious Diseases, Pediatric Clinical Research Office, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A. Morven S. Edwards Section of Infectious Diseases, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, U.S.A. R. Eggers Department of Immunizations, Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland Dubravka Grdic Eliasson Gothenburg, Sweden

Institute of Biomedicine, University of Gothenburg,

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Contributors

Jose´ Esparza

Bill & Melinda Gates Foundation, Seattle, Washington, U.S.A. Department of Molecular Virology and Microbiology, Houston,

Mary K. Estes Texas, U.S.A.

Lydia A. Falk Center for Biologics Evaluation and Research, FDA, Rockville, Maryland, U.S.A. V. Fischetti Rockefeller University, New York, New York, U.S.A. Richard A. Flavell Department of Immunobiology, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, U.S.A. Ian H. Frazer Diamantina Institute for Cancer Immunology and Metabolic Medicine, The University of Queensland, Woolloongabba, Queensland, Australia Lucy C. Freytag Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana, U.S.A. Arthur M. Friedlander U.S. Army Medical Research Institute of Infectious Diseases, Frederick; and Uniformed Services University of the Health Sciences School of Medicine, Bethesda, Maryland, U.S.A. Wyeth Vaccines Research, Scientific and Clinical Affairs, Paris,

Bernard Fritzell France

James E. Galen Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Roger I. Glass Fogarty International Center, National Institutes of Health, Bethesda, Maryland, U.S.A. Intercell USA, Inc., Gaithersburg, Maryland, U.S.A.

Gregory M. Glenn David Goldblatt

Institute of Child Health, London, U.K.

Michel Goldman Institute for Medical Immunology, Universite´ Libre de Bruxelles, Charleroi, Belgium Michael F. Good

Queensland Institute of Medical Research, Queensland, Australia

Barney S. Graham Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Guido Grandi Michel Gre´co

Novartis Vaccines and Diagnostics, Research Center, Siena, Italy Independent Vaccine Expert, Formerly Aventis Pasteur, Lyon, France

Renata Grifantini

Externautics, Siena, Italy

Beatrix Grubeck-Loebenstein Immunology Division, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria Farshad Guirakhoo Marc Gurwith

Sanofi Pasteur, Marcy l’Etoile, France

PaxVax, San Diego, California, U.S.A.

Thomas L. Hale Department of Enteric Infections, Division of Communicable Disease and Immunology, Walter Reed Army Medical Center, Washington, D.C., U.S.A. Neal A. Halsey Department of International Health, Institute for Vaccine Safety, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A. Christopher D. Hammerbeck United States Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland, U.S.A.

Contributors

Leonard C. Harrison Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, Burnet Clinical Research Unit, Parkville, Victoria, The Royal Melbourne Hospital, Parkville, Victoria, Australia Dietmar Herndler-Brandstetter Immunology Division, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria Luc Hessel

Policy Affairs, Europe, Sanofi Pasteur MSD, Lyon, France London School of Hygiene and Tropical Medicine, London, U.K.

David L. Heymann Adrian V. S. Hill

The Jenner Institute, University of Oxford, Oxford, U.K.

Stephen L. Hoffman

Sanaria Inc., Rockville, Maryland, U.S.A.

Susan K. Hollingshead Department of Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Jan Holmgren University of Gothenburg Vaccine Research Institute (GUVAX) and Department of Microbiology & Immunology, University of Gothenburg, Gothenburg, Sweden Jay W. Hooper United States Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland, U.S.A. Marcus A. Horwitz Department of Medicine, University of California Los Angeles, Los Angeles, California, U.S.A. Peter Hotez Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A. Michael Houghton

Epiphany Biosciences, Inc., San Francisco, California, U.S.A.

Christopher D. Huston Department of Medicine, The University of Vermont College of Medicine, Burlington, Vermont, U.S.A. Stuart N. Isaacs Department of Medicine, Division of Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. David C. Jackson Department of Microbiology & Immunology, The University of Melbourne, Parkville, Victoria, Australia Lisa Jacobs

GAVI Secretariat, Geneva, Switzerland

Suresh Jadhav

Serum Institute of India Ltd., Pune, India

Kathrin U. Jansen SVP Early Phase Programs, Wyeth Vaccine Research, Pearl River, New York, U.S.A. Robert E. Johnston Carolina Vaccine Institute, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A. James B. Kaper Department of Microbiology and Immunology, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Ruth Karron Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, U.S.A. David C. Kaslow

Merck, North Wales, Pennsylvania, U.S.A.

Stefan H. E. Kaufmann Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany Wendy Keitel Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A.

xxi

xxii

Contributors

Richard T. Kenney Clinical R&D and Medical Affairs, Vaccines for Virus Diseases, North America GlaxoSmithKline Biologicals, Columbia, Maryland, U.S.A. Dan E. Keyler Department of Medicine, Hennepin County Medical Center and College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, U.S.A. David M. Koelle

University of Washington, Seattle, Washington, U.S.A.

Hye-won Koo Pharmaco-Economics and Health Outcomes Research, Medical Department, GlaxoSmithKline Korea, Seoul, Korea Thomas R. Kosten Department of Psychiatry and Neuroscience, Baylor College of Medicine, Michael DeBakey Veterans Affairs Medical Center, Houston, Texas, U.S.A. Karen L. Kotloff Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Rosanna Lagos Centro para Vacunas en Desarrollo-Chile, Hospital de Nin˜os Roberto del Rio, Servicio de Salud Metropolitano Norte, Area Norte, Santiago, Chile Paul-Henri Lambert Centre of Vaccinology and Department of Pathology, University of Geneva, Geneva, Switzerland David E. Lanar U.S. Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, Silver Spring, Maryland, U.S.A. Claudio F. Lanata

Instituto de Investigacio´n Nutricional La Molina, Lima, Peru

Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Orin S. Levine Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A. George Lin

Biological Mimetics, Inc., Frederick, Maryland, U.S.A.

F.-Y. Lin Program of Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. Stephen F. Little U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, U.S.A. Margaret A. Liu ProTherImmune, Lafayette, California, U.S.A.; and Karolinska Institutet, Stockholm, Sweden J. Lloyd France

Program for Appropriate Technology in Health (PATH), Ferney-Voltaire,

Scott A. Lloyd Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Alex Loukas

Queensland Institute of Medical Research, Queensland, Australia

Igor S. Lukashevich Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Viviana P. Lutzky The EBV Biology Laboratory, Division of Infectious Diseases and Immunology, The Queensland Institute of Medical Research, Queensland, Australia Nils Lycke Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden Laura B. Martin

Novartis Vaccines Institute for Global Health, Siena, Italy

Contributors

Hugh S. Mason U.S.A.

The Biodesign Institute, Arizona State University, Tempe, Arizona,

Colin L. Masters Centre for Neuroscience, University of Melbourne, The Mental Health Research Institute, Victoria, Australia J. McCarthy

Queensland Institute of Medical Research, Queensland, Australia

Donald P. McManus Queensland Institute of Medical Research, Queensland, Australia Andrew McMichael Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, U.K. Helen McShane

The Jenner Institute, University of Oxford, Oxford, U.K.

B. Melgaard Department of Vaccines and Biologicals, World Health Organization, Geneva, Switzerland Mark L. Messonier National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Karen Midthun Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, U.S.A. Elizabeth Miller Immunisation Department, Centre for Infections, Health Protection Agency, London, U.K. Julie Milstien Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Dominique Missiakas Department of Microbiology, University of Chicago, Chicago, Illinois, U.S.A. Farrokh Modabber Drugs for Neglected Diseases Initiative (DNDi), Geneva Switzerland; Centre for Research and Training in Skin Diseases and Leprosy (CRTSDL), Tehran University of Medical Sciences, Tehran, Iran, & London School of Hygiene and Tropical Medicine, London, U.K. Thomas P. Monath Kleiner Perkins Caufield & Byers, Pandemic and Biodefense Fund, Menlo Park, California, U.S.A. Tsafrir Mor U.S.A.

The Biodesign Institute, Arizona State University, Tempe, Arizona,

Bernard Moss Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Denis J. Moss The EBV Biology Laboratory, Division of Infectious Diseases and Immunology, The Queensland Institute of Medical Research, Queensland, Australia E. Richard Moxon Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Children’s Hospital, Oxford, U.K. Peter Moyle School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia Brian R. Murphy Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, U.S.A. Gary J. Nabel Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

xxiii

xxiv

Contributors

Abdollah Naficy Parasitology and International Programs Branch, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Peter L. Nara Biological Mimetics, Inc., Frederick, Maryland, and Department of Biological Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa, U.S.A. Michel C. Nussenzweig Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, U.S.A. Derek T. O’Hagan Novartis Vaccines and Diagnostics, Inc., Cambridge, Massachusetts, U.S.A. Paul A. Offit Department of Pediatrics, Division of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Janet S. Ottinger Departments of Surgery and Immunology and Duke Translational Research Institute Immune Monitoring Core, Duke University Medical Center, Durham, North Carolina, U.S.A. Lawrence C. Paoletti Brigham and Womens’ Hospital, Harvard Medical School, Channing Laboratory, Boston, Massachusetts, U.S.A. Umesh D. Parashar National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Drew Pardoll Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Marcela F. Pasetti Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. James C. Paton School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia Paul R. Pentel Department of Medicine, Hennepin County Medical Center, University of Minnesota Medical School, Minneapolis, Minnesota, U.S.A. William A. Petri, Jr. Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, Virginia, U.S.A. Alberto Pinzon-Charry Australia

Queensland Institute of Medical Research, Queensland,

Stanley A. Plotkin Wistar Institute of Anatomy and Biology, University of Pennsylvania, Doylestown, Pennsylvania, U.S.A. Christopher V. Plowe Howard Hughes Medical Institute and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Andrew J. Pollard Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Children’s Hospital, Oxford, U.K. Jeff Powell VaxInnate Corporation, New Haven, Connecticut, U.S.A. Konstantin V. Pugachev Bali Pulendran U.S.A.

Sanofi Pasteur, Cambridge, Massachusetts, U.S.A.

Department of Pathology, Emory Vaccine Center, Atlanta, Georgia,

Karina Ramirez Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Mary Ramsay Immunisation Department, Centre for Infections, Health Protection Agency, London, U.K.

Contributors

Malla R. Rao International Tropical Diseases Research/Epidemiological Studies, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, U.S.A. Rino Rappuoli

Novartis Vaccines and Diagnostics S.R.L., Siena, Italy

Steven G. Reed

Infectious Disease Research Institute, Seattle, Washington, U.S.A.

Ana M. H. Restrepo Geneva, Switzerland

Initiative for Vaccine Research, World Health Organization,

Thomas L. Richie U.S. Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, Silver Spring, Maryland, U.S.A. Adam Richman

Sanaria Inc., Rockville, Maryland, U.S.A.

John B. Robbins Program of Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. Marjorie Robert-Guroff Vaccine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A. Roy M. Robins-Browne Department of Microbiology and Immunology, University of Melbourne, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia Sandra Romero-Steiner Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Maria S. Salvato Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Philippe Sansonetti Unite´ de Pathoge´nie Microbienne Mole´culaire, Institut Pasteur, Paris, France Robert W. Sauerwein Department of Medical Microbiology, Radbound University Nijmegen Medical Center, Nijmegen, The Netherlands Anne Schaap-Nutt Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, U.S.A. Alexander Schmidt Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, U.S.A. Rachel Schneerson Program of Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. Olaf Schneewind Illinois, U.S.A.

Department of Microbiology, University of Chicago, Chicago,

Angelika Schwanninger Immunology Division, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria Benjamin Schwartz National Vaccine Program Office, U.S. Department of Health and Human Services, Washington, D.C., U.S.A. J. Anthony G. Scott KEMRI Wellcome Trust Research Programme, Kilifi, Kenya; and Nuffield Department of Clinical Medicine, University of Oxford, Oxford, U.K. Robert C. Seid, Jr.

Intercell USA, Inc., Gaithersburg, Maryland, U.S.A.

John W. Shiver Department of Vaccines and Biologics Research, Merck Research Laboratories, Merck and Company, West Point, Pennsylvania, U.S.A.

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Contributors

Claire-Anne Siegrist WHO Collaborating Centre for Neonatal Vaccinology, Departments of Pediatric and Pathology and Immunology, University of Geneva, Geneva, Switzerland B. Kim Lee Sim

Sanaria Inc., Rockville, Maryland, U.S.A.

Jakub K. Simon Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Samba O. Sow Bamako, Mali

Centre pour le De´veloppement des Vaccins-Mali (CVD-Mali),

Lawrence R. Stanberry Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, New York, U.S.A. Ralph M. Steinman Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York, U.S.A. Iain Stephenson

Infectious Diseases Unit, Leicester Royal Infirmary, Leicester, U.K.

Peter C. Stowe Wyeth Biotech, Division of Wyeth Pharmaceuticals, Collegeville, Pennsylvania, U.S.A. Kanta Subbarao Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Alec Sutherland Arizona, U.S.A.

The Biodesign Institute, Arizona State University, Tempe,

Ann-Mari Svennerholm Gothenburg University Vaccine Research Institute and Department of Microbiology and Immunology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden Marcelo B. Sztein Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Shousun C. Szu Program of Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A. Carol O. Tacket Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Milagritos D. Tapia Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Sharon Tennant Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Istvan Toth School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia John Treanor Division of Infectious Diseases, Department of Medicine, University of Rochester Medical Center, Rochester, New York, U.S.A. Dennis W. Trent

Xcellerex, Inc., Marlborough, Massachusetts, U.S.A.

Christine Trumpfheller Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York, U.S.A. Jeffrey B. Ulmer U.S.A. David W. Vaughn U.S.A.

Novartis Vaccines and Diagnostics, Cambridge, Massachusetts, GlaxoSmithKline Biologicals, King of Prussia, Pennsylvania,

Contributors

Richard L. Ward Division of Infectious Diseases, Children’s Hospital Medical Center, Cincinnati, Ohio, U.S.A. Juliane Bubeck Wardenburg Departments of Pediatrics and Microbiology, University of Chicago, Chicago, Illinois, U.S.A. Charles Weijer Department of Philosophy, University of Western Ontario, London, Ontario, Canada Kent J. Weinhold Departments of Surgery and Immunology and Duke Translational Research Institute Immune Monitoring Core, Duke University Medical Center, Durham, North Carolina, U.S.A. Bruce G. Weniger National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. Piers Whitehead

Corporate Development, Neovacs SA, Paris, France

Stephen S. Whitehead Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. Richard J. Whitley Department of Pediatrics, Microbiology, Medicine, and Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A. Cynthia G. Whitney Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A. David Wood Department of Immunization, Vaccines and Biologicals, Quality, Standards and Safety Team, World Health Organization, Geneva, Switzerland J. Zabriski

Rockefeller University, New York, New York, U.S.A.

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1 Vaccines and Vaccination in Historical Perspective Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Rosanna Lagos

Centro para Vacunas en Desarrollo-Chile, Hospital de Nin~os Roberto del Rio, Servicio de Salud Metropolitano Norte, Area Norte, Santiago, Chile

Jose´ Esparza Bill & Melinda Gates Foundation, Seattle, Washington, U.S.A.

INTRODUCTION The history of immunization, from earliest attempts to modem genetically engineered vaccine candidates, represents a long road marked with many milestones. Extensive historical reviews document many of these cardinal achievements (1–4). A few of the most pivotal milestones are mentioned briefly in this chapter, such as variolation, Jenner’s experiments of inoculating subjects with cowpox to prevent smallpox, and the earliest live and inactivated bacterial and viral vaccines and toxoids. On the other hand, the main purpose of this chapter is to emphasize historical accounts of several aspects of vaccinology that are not generally well described. These include some early attempts at eliciting local immunity by means of oral vaccines, attempts over the centuries at grappling with the problem of how to assess the safety and efficacy of candidate vaccines before their widespread use, and the evolution of controlled field trial methodology.

THE DAWN OF IMMUNOPROPHYLAXIS The first attempts to prevent an infectious disease by means of immunoprophylaxis involved the process of ‘‘inoculation of the smallpox’’ or ‘‘variolation,’’ wherein the contents of smallpox vesicles, pustules, or scabs were used to inoculate individuals who had not previously experienced the disease (4). Records of this procedure date to about AD 1000 in China (4). Scabs from mildly affected smallpox patients were stored for approximately one month (longer in winter), ground up in a ratio of 4:1 with the plant Uvularia grandiflora, and then inoculated intranasally. A slight fever was expected six days thereafter, which rose markedly on the seventh day, to be followed by the onset of the rash on the ninth or tenth day following inoculation. Fatalities were reportedly uncommon compared with victims of natural smallpox infection. It was stated, ‘‘Not one in 10, not one in 100 does not recover’’ (4). Parenteral variolation was practiced in the Indian subcontinent, southwest Asia, and North Africa in the 16th and 17th centuries. Reports of variolation reached England as early as 1700 through letters to the Royal Society sent by Joseph

Lister, an Englishman working in China with the East India Company (5). Over the next 15 years, further reports came from many sources, and in 1713 the Greek physician Emmanuel Timonis published the first European article about variolation (6). Many references credit Lady Mary Wortley Montagu with having introduced the practice of variolation into Great Britain in 1721 (7). Lady Mary herself suffered from smallpox in 1715, leaving her pockmarked. While living in Constantinople as the wife of the British ambassador, she became aware of variolation, as it was practiced every autumn by skilled Turkish women. In 1718, Lady Montagu had her five-year-old son inoculated with smallpox under the supervision of Charles Maitland, the surgeon to the British Embassy (5,8). Lady Montagu wrote to a friend in England, Sarah Criswell, extolling the practice of variolation and vowed to make the procedure fashionable in England upon her return. In 1721, three years after she returned to England, an epidemic of smallpox raged in London. Lady Mary contacted Maitland, who was also in Great Britain at this time, and convinced him to variolate her four-year-old daughter. Maitland agreed, but demanded that there be two witnesses, one of whom was Dr James Keith (8). Keith was so impressed with the outcome that he had his six-yearold son variolated. Because these first inoculations were done in the face of considerable attention by the College of Physicians as well as the Royal Court, Lady Mary, whose insistence led to the first inoculation, has been widely credited with having introduced the practice into Great Britain. However, Miller (8) argues that Lady Mary’s contribution to variolation becoming an accepted and widespread practice in England was, in fact, quite minimal and that the real driving force for the introduction of variolation into the British Isles was Hans Sloane, physician to the king of England and president of the Royal Society. Zabdiel Boylston used variolation for the first time in the United States, during a smallpox epidemic in Boston in 1721. The new procedure was promoted by the clergyman Cotton Mather who, reportedly, learned it from Onesimus, one of his African slaves (9). In Latin America, variolation was probably first introduced in Chile in 1765 by another clergyman, Father Pedro Manuel Chaparro.

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During the last decades of the 18th century, smallpox was rampant in Europe, despite the increasing use of variolation as a preventive measure. Among rural folk during this period, it was increasingly appreciated that milkmaids were selectively spared the ravages of smallpox and that this was somehow related to the mild pox infection they often acquired from the cows they milked (2,4). Although several scholars and physicians in the period 1765 to 1791 acknowledged this association and some, like the farmer Benjamin Jesty, even inoculated family members with cowpox (2), appropriate credit must be given to Edward Jenner for his pioneer achievements (Fig. 1). In 1796, Jenner undertook to test rigorously the putative protective effect of a prior cowpox infection against smallpox by

actively immunizing an eight-year-old boy with cowpox and later challenging the child with smallpox (i.e., by variolation). Other vaccinations (or inoculation of the cowpox) of additional subjects, followed by smallpox challenge (variolation), were carried out thereafter. Jenner had the foresight and perseverance to publish his results and, for the rest of his professional life, promulgated the practice of ‘‘vaccine inoculation’’ (10,11). During the 19th century, smallpox vaccination became increasingly popular and accepted in other areas, including Europe and North America (4). A remarkable and often forgotten global public health campaign was the Royal Philanthropic Expedition of the Vaccine commissioned by King Charles IV of Spain and directed by the physicians Francisco Xavier de Balmis and Jose´ Salvany, that between 1802 and 1806 took the smallpox vaccine to Spain’s territories in the Americas and the Philippines (12,13). It is fitting that smallpox became the first (and so far the only) communicable disease to be actively eradicated, an accomplishment achieved in the decade 1967 to 1977. An enigma that remains unresolved after the eradication of smallpox concerns the origin of vaccinia, the smallpox vaccine virus. Whatever its origin, vaccinia is a separate species within Orthopoxvirus genetically distinct from both cowpox and variola viruses. Cowpox is in fact a rodent virus that occasionally infects other mammalian hosts (14). Hypotheses that have been promulgated include that it represents a hybrid between cowpox and variola virus, that it derives from cowpox virus, or that it is a descendant of a virus (perhaps of equine hosts) that

Figure 1 Edward Jenner (1749–1823), the father of vaccinology. An 1800 pastel portrait of Edward Jenner by J. R. Smith. Source: Photo courtesy of The Wellcome Institute Library, London, U.K.

Figure 2 Louis Pasteur (1822–1895), a 19th century pioneer of vaccinology. Source: Photo courtesy of Institute Pasteur, Paris, France.

It is important to emphasize that although variolation was a useful public health intervention at the time, it was not without risks. Early variolation procedures led to death in approximately 1% to 2% of the immunized subjects. This was considered an acceptable risk in view of the much higher likelihood of a fatal outcome if smallpox (a common disease at the time) were to be acquired by the natural mode of transmission. The other major drawback was that variolated subjects could themselves spread the smallpox virus to susceptible contacts. So it was clear that the control of smallpox needed a better preventive intervention.

THE ORIGIN OF VACCINATION

Chapter 1: Vaccines and Vaccination in Historical Perspective

no longer exists in nature. The recent sequencing of the genome of horsepoxvirus seems to support the concept that the vaccinia virus may indeed have been derived from horsepox, an origin that Jenner himself suspected (15). As Jenner demonstrated with cowpox (or horsepox) and smallpox, there are instances among viruses where, because of the host specificity of virulence, an animal virus gives aborted and attenuated infection in the human host, sometimes leading to an acceptable level of protection. Examples include influenza viruses, rotaviruses, and parainfluenza viruses. Remarkable is the fact that the Jennerian approach to immunoprophylaxis remains valid in modern vaccine development, as the reader will see in the chapter ‘‘Vaccines against Rotavirus Gastroenteritis.’’ Finally, it is worth noting that Louis Pasteur (Fig. 2), himself one of the most influential pioneers of vaccinology, coined the term ‘‘vaccine,’’ in honor of Jenner, to refer generically to immunizing agents.

THE FIRST USES OF ATTENUATED BACTERIA AS PARENTERAL AND ORAL IMMUNIZING AGENTS In the last quarter of the 19th century bacteriology became a burgeoning science. One after another, bacteria came to be revealed as etiological agents of important human diseases such as cholera, typhoid fever, plague, diphtheria, and tuberculosis and of veterinary diseases such as anthrax and tuberculosis. The ability to obtain pure cultures of the causative bacteria paved the way for the development of vaccines. Pasteur observed that cultures of Pasteurella septica, which causes the lethal disease fowl cholera in chickens, lost their virulence when the cultures were allowed to sit for two weeks (16). He found that chickens inoculated with the old cultures did not develop illness and, furthermore, were protected when subsequently inoculated with highly virulent fresh cultures. Pasteur concluded that in old cultures the bacteria undergo certain changes that result in attenuation but not in loss of immunogenicity. Pasteur applied his theory in an attempt to attenuate Bacillus anthracis, the cause of anthrax, an infection of cows, sheep, and goats (and occasionally humans) that is often fatal. Pasteur found that maintaining shallow cultures of B. anthracis at a temperature of 428C to 438C for two weeks resulted in a loss of virulence (furthermore, spores did not form at this temperature). In these early experiments, Pasteur established an approach that was followed in multiple later attempts at immunoprophylaxis: the first inoculations given in the immunization schedule were highly attenuated to maximize safety, whereas subsequent inoculations were somewhat less attenuated to increase antigenicity. On May 5, 1881, Pasteur and his colleagues Emile Roux and Charles Chamberland carried out a historic public experiment in Pouilly-le-Fort, France (17,18). They inoculated one set of farm animals (24 sheep, 1 goat, and 6 cows) with an initial highly attenuated vaccine; a second inoculation with a less-attenuated vaccine preparation was given on May 17. On May 31, these immunized animals and a set of uninoculated controls (24 sheep, 1 goat, and 4 cows) were challenged with virulent B. anthracis. Over the next four days, spectacular results documented the efficacy of the vaccine: in the control group, the 24 sheep and the goat died and the four cows became overtly ill, whereas there was only one death (a sheep) among the vaccinated animals. Within a short time, the anthrax vaccine became widely used in France. As early as 1882, Pasteur was able to report excellent

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results from the use of the vaccine in more than 79,000 sheep in France (19). The first bacterial vaccine used in humans was administered in 1884, barely one year after the initial isolation of Vibrio cholerae by Robert Koch (20), when Jaime Ferran inoculated live, putatively weakened, V. cholerae parenterally (21). Ferran’s vaccine, which consisted of broth cultures containing ‘‘attenuated’’ vibrios, was given to about 30,000 individuals who eagerly sought protection during the 1884 epidemic of cholera in Spain. This experience generated much interest internationally, and commissions from several countries came to inspect and evaluate Ferran’s work. The most influential committee, sponsored by the Pasteur Institute, Paris, criticized Ferran’s vaccine and argued that no convincing proof was provided to support claims for a prophylactic effect (22). Furthermore, it was reported that Ferran’s live vaccine was heavily contaminated with other microorganisms and that only a small proportion of the bacteria were V. cholerae (22); contamination may have accounted for the severe adverse reactions associated with this vaccine and its apparent lack of efficacy (22). In 1891, only one year after Waldemar Haffkine had joined the Pasteur Institute in Paris, Pasteur asked him to carry out research to develop an immunizing agent against cholera (23). Following Pasteur’s general principle that live vaccines confer protection superior to vaccines consisting of killed microorganisms, Haffkine prepared two modified V. cholerae strains for use as live vaccines. The first strain was attenuated by culture at 398C with continuous aeration, whereas the second strain underwent multiple intraperitoneal passages in guinea pigs in an attempt to increase its virulence. Haffkine utilized these strains sequentially as parenteral immunizing agents; the attenuated strain was inoculated first, followed six days later by the strain of supposedly enhanced virulence (24). Typical side reactions following vaccination included fever, malaise, and headache as well as pain and swelling at the injection site. In later evaluations of the vaccine, Haffkine abandoned the initial inoculation with the attenuated vibrio and administered to humans only the pathogenic strain without an increase in adverse reactions. Statistical analysis of several clinical trials of Haffkine’s live cholera vaccine in India suggested that it was efficacious (25). Nevertheless, further use of the vaccine was abandoned because of difficulty in standardizing it and producing it in large quantities. The other early success in attenuated bacterial vaccines was the bacille Calmette-Guerin (BCG) vaccine against tuberculosis. Leon Charles Albert Calmette and Jean-Marie Camille Guerin obtained a stable, attenuated strain incapable of causing tuberculosis in the highly susceptible guinea pig (26). It was achieved by repeatedly subculturing (213 times over 13 years), in the presence of ox bile, a tubercle bacillus originally isolated from a cow by Edmond Nocard in 1902 (26). The first administration of BCG vaccine to a human occurred in 1921, when a newborn infant, whose mother had died of tuberculosis, was given an oral dose without adverse effects. Calmette initially advised that the vaccine should be administered orally to young infants. Accordingly, by the late 1920s, approximately 50,000 French infants had received the apparently well-tolerated BCG vaccine (27). By the late 1920s, the intradermal route of inoculation rapidly began to replace the oral route of vaccination. It was not until the 1950s that controlled field trials confirmed the efficacy of at least one strain of BCG (28).

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EARLY INACTIVATED WHOLE-CELL BACTERIAL VACCINES Three parenteral inactivated whole-cell bacterial vaccines to protect humans against cholera, typhoid fever, and plague, originally developed at the end of the 19th century, were used with little modification for three quarters of a century thereafter. In each instance, the isolation of the causative agent in pure culture was followed shortly thereafter by the development of vaccine candidates.

Cholera Vaccines In 1896, Wilhelm Kolle (29) recommended the use of agargrown, heat-inactivated whole V. cholerae organisms as a parenteral immunizing agent. This nonliving vaccine was markedly simpler to prepare and to standardize than Haffkine’s live parenteral vaccine. By 1911, Haffkine was also utilizing inactivated vibrios as a vaccine with 0.5% phenol as a preservative (30). Kolle-type vaccines were first used on a large scale during the 1902 cholera epidemic in Japan (31). In the 1960s and early 1970s, randomized, controlled field trials carried out in Bangladesh (32–34), the Philippines (35), Indonesia (36), and India (37) documented that killed whole-cell cholera vaccines can confer significant short-term protection in older children and adults.

Typhoid Vaccines In 1896, Richard Pfeiffer (38) and Almroth Wright (39) independently reported that a vaccine against typhoid fever could be prepared by inactivating cultures of typhoid bacilli with heat and preserving them in phenol (40). By 1915, killed whole-cell parenteral typhoid vaccines had become widely used by the military in Europe and the United States. Systematic use of the vaccine in the U.S. army, in 1912, was followed by a diminution of approximately 90% in the incidence of typhoid fever (41). Thus, epidemiological data suggested that the vaccine was protective (42), although rigorous controlled field trials of efficacy of the parenteral heat-phenol typhoid vaccine were not carried out until the 1950s (43). Controlled field trials sponsored by the World Health Organization (WHO) in the 1950s and 1960s demonstrated that the heat-phenolized vaccine conferred about 50% to 75% protection against typhoid fever (42,44–46).

final inoculation was with minimally dried cord, which contained virulent virus. Needless to say, this vaccination procedure was quite controversial, even among the members of Pasteur’s group. What is extraordinary is how Pasteur and colleagues identified the tissues wherein the rabies virus resides and how they managed to achieve attenuation yet retain immunogenicity. In the late 1920s and early 1930s (49,50), Max Theiler, a South African physician working at the Rockefeller Foundation, developed an attenuated strain of yellow fever virus by repeated passage of the wild-type Asibi strain in minced chick embryo tissue from which the head and spinal cord had been selectively removed to minimize the amount of nerve tissue. Somewhere between the 89th and 114th passages, the virus lost its neurotoxicity. Theiler adapted this attenuated virus, strain 17D, to grow in chick embryos. In the 1930s and 1940s, this attenuated virus vaccine set a standard for safety, immunogenicity, and efficacy that continues to draw admiration today. Strain 17D remains one of the best all-around vaccines ever developed. It has been safely given to hundreds of millions of adults and children and provides long-term protection. This is an amazing feat, but particularly so when one considers that the vaccine was developed in an era before modem tissue culture techniques and concepts of viral genetics had evolved. Max Theiler received the Nobel Prize in 1951.

‘‘SUBUNIT’’ AND ‘‘EXTRACT’’ VACCINES The early diphtheria and tetanus toxoids should be regarded as the pioneer subunit vaccines. In each instance their development followed a similar course. Discovery of the etiological agents of diphtheria by Edwin Klebs (51) and Frederick Loeffler (52) and of tetanus by Shibasaburo Kitasato (53) was followed by the demonstration that Corynebacterium diphtheriae and Clostridium tetani elaborate potent exotoxins. Inoculation of broth cultures of these bacteria through porcelain filters resulted in sterile filtrates that were toxic for animals, leading to syndromes characteristic of human disease (54). The production in horses of specific antitoxins for passive protection and treatment came next (Fig. 3), and this was followed by the

LIVE VIRUS VACCINES Following their success with the development of a vaccine against anthrax, Pasteur and coworkers turned their attention toward the problem of rabies (47,48). Although unable to cultivate the virus as they could a bacterium, they nevertheless established that the infectious agent resided within the spinal cord and brain of infected animals. Pasteur and his team inoculated nerve tissue from a rabid animal submeningeally into rabbits and removed the spinal cord after the rabbits died; they were able to pass the infection from rabbit to rabbit in this manner. Roux discovered that if the spinal cords were desiccated for 15 days, they lost their ability to induce rabies. Spinal cords dried for fewer than two weeks were less attenuated, whereas minimally dried spinal cord clearly contained virulent virus. Pasteur’s group prepared a vaccine that consisted of dried spinal cord suspended in saline. Their immunization schedule involved daily inoculations for 14 days, commencing with material from spinal cord that had been dried for 14 days and progressing on the successive days to the use of cord dried for less and less time. This was continued until, after two weeks, the

Figure 3 An immunized horse being bled for serum as a source of diphtheria antitoxin at the CSL, Parkville, Victoria, Australia, 1920. Abbreviation: CSL, Commonwealth Serum Laboratories. Source: Photo courtesy of CSL.

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Figure 4 Diphtheria toxoid production at the CSL, Parkville, Victoria, Australia, 1929. Abbreviation: CSL, Commonwealth Serum Laboratories. Source: Photo courtesy of CSL.

use of toxin/antitoxin mixtures to achieve active immunization (55). As will be reviewed later, mistakes in the preparation of such mixtures sometimes led to disastrous consequences. Ultimately, it was found that by treatment with formalin, diphtheria and tetanus toxins could be rendered biologically innocuous yet retain their ability to stimulate neutralizing antitoxin (56,57). Alexander Glenny (57) claims to have prepared formalinized diphtheria toxoid as early as 1904. In a personal communication to Henry Parish (1), Glenny related that his first toxoid was prepared by accident! He observed that a batch of diphtheria toxin lacked toxic activity yet elicited antitoxin in animals as efficiently as fully active toxin. The C. diphtheriae cultures used to produce the toxin had been grown in large earthenware containers that could not be readily sterilized. One of the steps in sterilizing the containers for the next batch involved washing them with formalin. Glenny hypothesized that residual formalin had apparently inactivated the toxin. He subsequently proved that formalin could, indeed, alter diphtheria toxin to toxoid, rendering it innocuous yet preserving its antigenicity (Fig. 4).

STIMULATION OF LOCAL IMMUNITY Many infectious agents interact with the mucosa of the gastrointestinal tract, the respiratory tract, or the urinary tract as a site of colonization or as a preliminary step before invasion. Recognition of the mucosal immune system as a unique component of the overall immune system of the mammalian host underlies extensive current research to develop oral or intranasal vaccines to prevent enteric infections such as cholera, typhoid fever, rotavirus diarrhea, and shigellosis and respiratory infections such as influenza and RSV bronchiolitis. The leading pioneer in the concept of local immunization was Alexandre Besredka (58), who was generations ahead of his time in his approach and his concepts. Besredka, however, did not believe that antibodies were involved in mediating the local immunity that he stimulated. Albert B. Sabin’s pioneering work resulting

Figure 5 Albert B. Sabin (1906–1993), a 20th century pioneer of vaccinology, administering oral polio vaccine in 1959. Source: Photo courtesy of Heloisa Sabin.

in a practical and effective live oral vaccine against poliomyelitis set a paradigm for other oral and nasal vaccines (Fig. 5). The parts of this book describing vaccines against cholera show that the modem approach to prevention of this disease involves oral immunization with either inactivated antigens or attenuated bacteria. However, these modern oral cholera vaccines are descendants of a long tradition. For example, the first report of nonliving whole V. cholerae used as an oral vaccine in humans was published in 1893 (59); this report related the lack of adverse reactions following ingestion of multiple doses containing billions of inactivated vibrios. In the 1920s and 1930s, field trials of Besredka’s killed oral V. cholerae vaccine, combined with bile (so called ‘‘bilivaccine’’), were carried out in India (60–62) and Indochina (63). Significant protection was apparently achieved. In the Indian trials, the oral vaccine was also compared with a killed parenteral whole-cell vaccine. However, it is not certain that the vaccine and control (nonvaccinated) groups were fairly randomized so that the risk of infection was equal. Nevertheless, the oral bilivaccine provided 82% protection and the parenteral vaccine 80% protection during the period of surveillance (60–62). The bilivaccine was administered in a total of three doses on consecutive days; the subject to be vaccinated first ingested a bile tablet, followed, 15 minutes thereafter, by a bilivaccine tablet containing 70 billion dried vibrios. Because of the bile component, the bilivaccine commonly caused adverse reactions, including nausea, vomiting, and acute diarrhea; it appears

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that, in large part as a consequence of these reactions, further work with the bilivaccine was abandoned. Early investigators who pursued the concept of coproantibodies in the gut and local antibodies on other mucosal surfaces include Arthur Davies in the 1920s (64); Torikata and Imaizuma (65), and Theodore Walsh and Paul Cannon in the 1930s (66); and William Burrows (67) in the 1940s.

CLINICAL EVALUATION OF THE SAFETY AND EFFICACY OF VACCINE CANDIDATES When a vaccine candidate appears, its safety must first be demonstrated in a series of small clinical trials before largescale use of the vaccine can be considered. The need for such studies was recognized as early as the introduction of variolation in England in 1722, when royal permission was given to variolate six condemned prisoners in an effort to determine the safety of that procedure (5,8). The prisoners were offered pardon in exchange for their participation, if they survived. In November 1721, it was announced in the London newspapers that some orphan children of St. James parish, Westminster, would be inoculated as an experiment to assess the effect of variolation in children (5,6). The use of prisoners and institutionalized children for vaccine safety studies in the 1720s established a precedent that continued until the mid-1970s. For example, in the United States in the 1950s, the attenuated poliovirus vaccine was initially tested for safety and immunogenicity in adult prison volunteers (68), whereas the inactivated poliomyelitis vaccine was evaluated early on in institutionalized children (69). In the early 1970s, the ethics of experimentation in such populations, particularly prisoners, underwent reevaluation (70). A new consensus emerged that considered prisons to be inherently coercive environments in which it was difficult to guarantee informed consent (71). As a consequence, by the mid-1970s, the use of prison volunteers for phase I studies to assess the safety and immunogenicity of vaccines had virtually disappeared. There also exists a long history of evaluating the efficacy of candidate vaccines in experimental challenge studies. In the first of Jenner’s famous experiments, James Phipps, an eightyear-old boy was inoculated on the arm with cowpox. Jenner wrote (11): ‘‘Not withstanding the resemblance which the pustule, thus excited on the boy’s arm, bore to variolous inoculation, yet as the indisposition attending it was barely perceptible, I could scarcely persuade myself the patient was secure from the Small Pox. However, on his being inoculated some months afterward, it proved that he was secure. This case inspired me with confidence; and as soon as I could again furnish myself with Virus from the Cow, I made an arrangement for a series of inoculations. A number of children were inoculated in succession, one from the other; and after several months had elapsed, they were exposed to the infection of the Small Pox; some by Inoculation, others by variolous effluvia, and some in both ways; but they all resisted it.’’ To put Jenner’s challenge of cowpox-vaccinated children with smallpox virus into proper perspective, one must appreciate that variolation was a widespread practice in England in the last quarter of the 18th century (72). In Berlin, Germany, during the Great Depression, Wolfgang Casper, a physician who worked in the gonococcal wards

of the Rudolf Virchow Hospital, developed a gonococcal vaccine consisting of a polysaccharide extract of gonococci (73). In 1930, he carried out an unusual clinical experiment to test its efficacy. Casper recruited 10 destitute individuals whom he had previously seen with gonorrhea and who had recovered (3). These 10 volunteers were moved into a ward at the hospital and provided with room and board. Five received his gonococcal vaccine and five were injected with a placebo. At a later point, a female volunteer, a prostitute, was brought onto the ward to spend one night with the 10 male volunteers, all of whom had sexual intercourse with the prostitute. Within one week, four of the five placebo recipients had developed gonorrhea versus none of the five recipients of Casper’s vaccine (3). Preliminary, small-scale clinical studies to assess the safety and immunogenicity of new candidate vaccines (phase I studies) constitute a critical first step in the evaluation of any vaccine, as is discussed in chapter 3. Experimental challenge studies to assess vaccine efficacy represent an important step in the development of vaccines for certain infectious agents that readily respond to antimicrobial therapy or that cause self-limited illness and for which wellestablished models of experimental infection exist. Data from these safety, immunogenicity, and challenge studies serve to identify vaccine candidates worthy of further evaluation in large-scale field trials. In recent years, this evaluation process has been applied to vaccines against cholera (74,75), shigellosis (76), malaria (77), and influenza (78). In the modem era, there exist strict ethical guidelines to recruit the volunteers who participate in such clinical trials and to obtain their informed consent, assuring that they understand the potential risks involved and the procedures to which they will be exposed. Under sponsorship of the WHO, the Vaccine Trial Centre was established at the Faculty of Tropical Medicine of Mahidol University in Bangkok, Thailand, in the mid-1980s. This represented the first unit in a developing country where challenge studies with various pathogenic organisms could be undertaken to assess vaccine efficacy following rigorous ethical and technical local review of protocols according to international standards (79). The relative importance of experimental challenge studies that assess vaccine efficacy in volunteers increased in 1993, when the Vaccines and Related Biologic Products Advisory Committee to the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration (FDA) voted that the results of such studies should constitute sufficient evidence of the vaccine’s efficacy for submission of a Product Licensure Application. The case in point considered by the committee was the efficacy of a new live oral cholera vaccine for use in adult U.S. travelers (80). The conclusion was that the challenge studies provided a better measure of the efficacy of the vaccine for U.S. adults than would the results of a field trial in an endemic area involving a population repeatedly exposed to cholera antigens. As an earlier precedent, the efficacy of live oral cholera vaccine CVD 103-HgR as demonstrated in volunteer challenge studies was sufficient to allow the licensure of that vaccine in Switzerland, Canada, and Australia.

LARGE-SCALE FIELD TRIALS TO ASSESS VACCINE EFFICACY In modem times, the prospective randomized, double-blind, controlled field trial under conditions of natural challenge is the definitive ‘‘gold standard’’ test of the efficacy of a vaccine. In

Chapter 1: Vaccines and Vaccination in Historical Perspective

general, the result of at least one such trial is required for licensure of a vaccine in most countries. One exception to this general rule has been cited above. Historically, the development of epidemiological methods for conducting field trials to evaluate the efficacy of vaccines represented an obvious and necessary offspring of the development of new vaccines themselves. According to Cvjetanovic (25), the first attempts to determine vaccine efficacy by controlled field trials were the tests of Haffkine’s live cholera vaccine in India. Initially, uncontrolled trials were carried out by Haffkine throughout India, in 1893 and 1894, involving 42,197 individuals, and, from 1895 through 1896, involving an additional 30,000 persons (81–83). However, the historic testing of his vaccine, from the epidemiological perspective, ‘‘involved relatively small groups of individuals residing in prisons and on tea plantations’’ (81–83). As reviewed by Cvjetanovic (25), Haffkine concluded that to properly assess the efficacy of his cholera vaccine, equal-sized groups of individuals should be compared, who were randomly allocated to receive vaccine or to serve as unimmunized controls and who were at essentially identical risk of exposure to natural infection. A rigorously designed and executed large-scale vaccine field trial that included a number of the features (albeit not all) of modem-controlled vaccine efficacy trials was the evaluation by Macleod et al. (84) of a multivalent pneumococcal polysaccharide vaccine carried out among U.S. recruits at a training base during World War II. In this double-blind study in a highrisk population, 7730 persons were allocated to receive vaccine or saline placebo and surveillance was maintained for pneumococcal disease with bacteriological confirmation of clinical cases. One decade after Macleod’s field trial of pneumococcal vaccine in several thousand participants, the famous Francis field trial of inactivated Salk poliovirus vaccine was undertaken in the United States, involving the inoculation of more than

Figure 6 Drs. Albert B. Sabin (left) and Jonas Salk conferring during a press conference that followed a congressional hearing (June 22, 1955) on a bill to allocate federal funds to support vaccination of U.S. children with the inactivated polio vaccine.

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650,000 children (85). The impeccably designed and executed Francis field trial constitutes a monument in the history of vaccine field trials. It incorporated all the fundamental features of modern field trial design and accomplished the logistics, data management, and data analysis in an era before computer technology, cellular telephones, and other technological aids were available. Moreover, the results of the field trial were so convincing that historic public health legislation ensued in the United States. Just a few months after announcement of the field trial results and subsequent congressional hearings (Fig. 6), the U.S. Congress passed legislation that provided financial assistance to the states in the form of grants to allow all children and pregnant women an opportunity to be vaccinated against poliomyelitis. Chapter 5 shows how much further field trial methodology has evolved from the 1940s and 1950s when Macleod and coworkers and Francis and coworkers, respectively, carried out their hallmark trials.

VACCINE CALAMITIES An occasional byproduct of the development of new vaccines has been the inadvertent occurrence of severe adverse reactions or fatalities because of contamination, incomplete attenuation, inadequate detoxification, or idiosyncrasy. Such untoward events were obviously more common with the early vaccines. They led to an awareness of the importance of maintaining strict procedures for manufacture, testing of safety (Fig. 7), potency, purity, and (where relevant) sterility. These events also gave rise to regulatory agencies to oversee the control of biological products. Sir Graham Wilson devoted an entire book, The Hazards of Immunization, to this topic (86); it contains material up to the mid-1960s. Some of the more prominent disasters and incidents related to vaccines, culled from various sources, are briefly summarized below.

Figure 7 Early animal safety test of a biological product, CSL, Parkville, Victoria, Australia. Abbreviation: CSL, Commonwealth Serum Laboratories. Source: Photo courtesy of CSL.

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EVENTS WITH VACCINES IN THE FIRST HALF OF THE 20TH CENTURY The Mulkowal Disaster In October 1902, in Mulkowal, India, 19 persons died from tetanus after being inoculated with Haffkine’s inactivated parenteral whole-cell plague vaccine drawn from the same bottle. An investigative commission concluded that contamination had occurred during manufacture of the vaccine in Haffkine’s laboratory in Bombay (Mumbai), India (23). Initially, Haffkine was held personally responsible and was suspended as director of the government laboratory at Bombay. Sir Ronald Ross was instrumental in releasing the report of a board of inquiry, showing that Haffkine and his laboratory were blameless (23). Under further pressure from Sir Ronald Ross, Haffkine was officially exonerated and offered the directorship of a laboratory in a government hospital in Calcutta (Kolkata). In a final vindication, in 1925, the Plague Research Laboratory of Bombay was renamed the Haffkine Institute.

The Lubeck Disaster When BCG vaccine was initially introduced, it was administered by the oral route and was given primarily to young infants. In Lubeck, Germany, approximately 250 infants were inadvertently fed virulent Mycobacterium tuberculosis instead of attenuated BCG (87,88); 72 of these infants died of tuberculosis, all but one within 12 months. The virulent strain of human tubercle bacillus had been kept in the same laboratory as the stock for the BCG vaccine and had inadvertently been used instead of the vaccine strain. Investigation of the incident vindicated the safety of the BCG vaccine and initiated regulatory measures to assure proper laboratory conditions, training of personnel, and procedures in laboratories where vaccines are manufactured.

Disasters Following Diphtheria Immunization As noted above, the earliest active immunization against diphtheria consisted of concomitant administration of mixtures of diphtheria toxin and antitoxin. Tragedies caused by efforts in manufacture were recorded in Dallas, Texas; Concord and Bridgewater, Massachusetts; Baden, Austria (86,89); Bundaberg, Russia (90); and China (91). In another instance, in Bundaberg, Australia, a diphtheria toxin-antitoxin mixture became contaminated with Staphylococcus aureus during manufacture of a product that contained no preservative (86,92). Of 21 children inoculated from the same bottle, 12 died of sepsis, 6 others became seriously ill but survived, while only 3 children remained healthy. S. aureus was isolated from abscesses in the ill but surviving children.

Typhoid Fever Following Immunization with a Heat-Treated Oral Typhoid Vaccine In 1904, bacteriologists in the U.S. army proposed to administer killed typhoid bacilli as an oral vaccine against typhoid fever. The bacterial culture was intended to be inactivated by heating at 568C for one hour. Initial cultures of the heated vaccine were sterile. Of 13 men who ingested the vaccine, 7 developed clinical typhoid fever and 3 others suffered ‘‘febrile illness,’’ with onsets 6 to 16 days after ingestion of the first dose of vaccine (93). Repeat bacteriological examination of the vaccine demonstrated that a few viable typhoid bacilli were recoverable (two to three organisms per milliliter).

Hepatitis Following Vaccination Against Yellow Fever Attenuated yellow fever virus vaccine strain 17D developed by Theiler remains one of the safest and most effective vaccines ever developed. However, during World War II, it was administered to U.S. servicemen along with human immune serum. Among approximately 2.5 million troops vaccinated, 28,600 cases of icteric hepatitis occurred, leading to 62 deaths (94); it is estimated that overall approximately 300,000 infections (mostly subclinical or non-icteric) may have occurred. By means of careful epidemiological investigations and volunteer studies, it was discovered that some lots of serum used as stabilizer were contaminated with a hepatitis virus (now known to have been hepatitis B) (95,96). When human serum ceased to be given along with the yellow fever vaccine, the problem disappeared.

SOME EVENTS IN THE LATTER HALF OF THE 20TH CENTURY The Cutter Incident Shortly after the favorable results of the Francis field trial of Salk, inactivated poliovirus vaccine were publicized in April 1955; the FDA licensed Salk-type vaccine prepared by several manufacturers. During a 10-day period in April 1955, a total of 120,000 children were immunized with two lots of inactivated vaccine manufactured by Cutter Laboratories of Berkeley, California (97–99). Cases of poliomyelitis occurred among 60 recipients of these vaccine lots and 89 of their family members. The median incubation period for vaccinees was eight days, whereas the median incubation was 24 days for the family contacts. During faulty production, wild poliovirus did not have sufficient contact with formalin to inactivate all the virus present.

Swine Influenza Vaccine and Guillain-Barre´ Syndrome In the United States in the spring of 1976, fatal influenza occurred in two individuals (one of whom was a healthy young adult) from whom a ‘‘swine’’ influenza virus (Hsw1Nl) was cultured. By serological studies, it was found that the virus antigenically resembled that of the great influenza epidemic of 1918 to 1919, which was characterized by high case fatality even in young adults. Accordingly, the U.S. Public Health Service, fearing a possible large-scale outbreak of Hsw1N1 disease in the coming winter, undertook a national program to prepare a swine influenza vaccine and to initiate a nationwide immunization campaign. The intention was to have the vaccine prepared and safety-tested and mass vaccination under way before the onset of the winter influenza season. Between October 1 and midDecember 1976, approximately 45 million doses of swine influenza vaccine were administered. However, beginning in late November and early December, reports began to appear of the occurrence of Guillain-Barre´ polyneuritis syndrome among recent recipients of vaccine. By December 16, the findings of a preliminary investigation corroborating this association led to a discontinuation of further immunizations with the vaccine. An extensive and detailed investigation carried out by prominent epidemiologists led by Alexander Langmuir (100) concluded that the administration of the swine influenza vaccine during the national campaign resulted in a 3.96- to 7.75-fold increase in risk of developing Guillain-Barre´ syndrome during the first six

Chapter 1: Vaccines and Vaccination in Historical Perspective

weeks post vaccination compared with the normal, expected, endemic incidence. Notably, Alexander Langmuir had also led the epidemiological investigation of the Cutter inactivated poliomyelitis vaccine incident two decades earlier (97–99).

SUMMARY COMMENT We find ourselves entering a particularly promising era in the history of vaccinology, spurred on by application of the tools of modem biotechnology that are resulting in a new generation of novel vaccines. And yet, even as we look ahead with great anticipation at this newly unfolding ‘‘golden era’’ of vaccinology, we glance back with admiration at the legacy of pioneering achievements left by our scientific forefathers, who developed the first vaccines, provided our initial understanding of immune mechanisms, and forged the early methods to assess the safety, immunogenicity, and efficacy of vaccines in clinical and field trials.

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18. Pasteur L, Chamberland C-E, Roux E. Classics of Biology and Medicine. Summary report of the experiments conducted at Pouilly-le-Fort, near Melun, on the Antrax vaccination. Yale J Biol Med 2002; 75:59–62. 19. Pasteur L. Une statistique au sujet de la vaccination pre´ventive contre le charbon, portant sur quatre vingt-cinq-mille animaux. CR Acad Sci Paris 1882; 95:1250–1252. 20. Koch R. Der zweite bericht der deutschen cholera commission. Dtsch Med Wochenschr 1883; 9:743–747. 21. Ferran J. Sur la prophylaxie du chole´ra au moyen d’injections hypodermiques de cultures pures du bacille-virgule. CR Acad Sci Paris 1885; 101:147–149. 22. Bornside GH. Jaime Ferran and preventive inoculation against cholera. Bull Hist Med 1981; 55:516–532. 23. Bomside GH. Waldemar Haffkine’s cholera vaccines and the Ferran-Haffkine priority dispute. J Hist Med Allied Sci 1982; 37:399–422. 24. Haffkine WM. Inoculation de vaccins antichole´riques a` l’homme. CR Seanc Soc Biol 1892; 44:740–746. 25. Cvjetanovic B. Contribution of Haffkine to the concept and practice of controlled field trials. Prog Drug Res 1975; 19:481–489. 26. Calmette A, Gue´rin C, Weill Halle B. Essais d’immunisation contre l’infection tuberculeuse. Bull Acad Med 1924; 91:787. 27. Calmette A. La vaccination preventive contre la tuberculose par le BCG. Paris: Masson, 1927. 28. Great Britain Medical Research Council. BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early life. Br Med J 1956; 1:413–427. 29. Kolle W. Zur aktiven Immunisierung des Menschen gegen Cholera. Zentralbl Bakteriol I Abt Orig 1896; 19:97–104. 30. Manifold CC. Report of a case of inoculation with carbolized anti-choleraic vaccine (Haffkine). Indian Med Gaz 1893; 28: 101–103. 31. Murata N. Uber die Schutzimpfung gegen Cholera. Zentralbl Bakteriol I Abt Orig 1904; 35:605. 32. Benenson AS, Mosely WH, Fahimuddin M, et al. Cholera vaccine field trials in East Pakistan: 2. Effectiveness in the field. Bull World Health Organ 1968; 38:359–372. 33. Mosley WH, Woodward WE, Aziz KMS, et al. The 1968–1969 cholera vaccine field trial in rural East Pakistan: effectiveness of monovalent Ogawa and Inaba vaccines and a purified Inaba antigen, with comparative results of serological and animal protection tests. J Infect Dis 1970; 121(suppl):SI–S9. 34. Mosley WH, Aziz KMS, Mizanur Rahman ASM, et al. Field trials of monovalent Ogawa and Inaba cholera vaccines in rural Bangladesh–three years of observation. Bull World Health Organ 1973; 49:381–387. 35. Philippines Cholera Committee. A controlled field trial of the effectiveness of cholera and cholera El Tor vaccines in the Philippines. Bull World Health Organ 1965; 32:603–625. 36. Saroso JS, Bahrawi W, Witjaksono H, et al. A controlled field trial of plain and aluminum hydroxide adsorbed cholera vaccines in Surabaya, Indonesia, during 1973–75. Bull World Health Organ 1978; 56:619–627. 37. Das Gupta A, Sinha R, Shrivastava DL, et al. Controlled field trial of the effectiveness of cholera and cholera El Tor vaccines in Calcutta. Bull World Health Organ 1967; 37:371–385. 38. Pfeiffer R, Kolle W. Experimentelle Untersuchungen zur Frage der Schutzimpfung des Menschen gegen Typhus abdominalis. Dtsch Med Wochenschr 1896; 22:735–737. 39. Wright AK. On the association of serous hemorrhages with conditions and defective blood coagulability. Lancet 1896; 2: 807–809. 40. Groschel DHM, Hornick RB. Who introduced typhoid vaccination: Almoth Wright or Richard Pfeiffer? Rev Infect Dis 1981; 3: 1251–1254. 41. Siler JF, Dunham GC, Longfellow D, et al. Immunization to typhoid fever. Section II. Historical review of the use of typhoid vaccine in the Army, Navy and Civilian Conservation Corps. Am J Hyg 1941; 17:7–38.

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68. Sabin AB. Behaviour of chimpanzee–avirulent poliomyelitis viruses in experimentally infected human volunteers. Am J Med Sci 1955; 230:1–8. 69. Salk JE, Bennett BL, Lewis LJ, et al. Studies in human subjects on active immunization against poliomyelitis: 1. A preliminary report of experiments in progress. JAMA 1953; 151:1081–1098. 70. Academy Forum. Experiments and Research with Humans: Values in Conflict. Washington, DC: National Academy of Sciences, 1975. 71. National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. Research involving prisoners. Appendix to report and recommendations. DHEW publication No. (OS) 76–132, 1976. 72. Kerns T. Jenner on Trial: The Ethics of Vaccine Research in the Age of Smallpox and the Age of AIDS. Lanham, Maryland: University Press of America, 1997:1–116. 73. Casper WA. Spezifische cutieaktionen an gonorrhoikern mit spezifischen, eiwess-freien substanzen aus gonokokken. Klin Wochenschr 1930; 9:2154–2158. 74. Levine MM, Kaper JB, Herrington F, et al. Safety, immunogenicity and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103 HgR. Lancet 1988; 2:467–470. 75. Black RE, Levine MM, Clements ML, et al. Protective efficacy in humans of killed whole-Vibrio oral cholera vaccine with and without the B subunit of cholera toxin. Infect Immun 1987; 55:1116–1120. 76. Herrington D, Van de Verg L, Formal SB, et al. Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease. Vaccine 1990; 8:353–357. 77. Herrington D, Clyde DF, Losonsky G, et al. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature 1987; 328:257–259. 78. Clements ML, Betts RF, Murphy BF, et al. Advantage of live attenuated cold-adapted influenza virus over inactivated vaccine for A/Washington/80 (H3N2) wild-type infection. Lancet 1984; 1: 705–708. 79. Sunthurasamai P, Migasena S, Vongsthongsri U, et al. Clinical and bacteriological studies of El Tor cholera after ingestion of known inocula in Thai volunteers. Vaccine 1992; 10:502–505. 80. Minutes of the Meeting of the Vaccines and Related Biologic Products Advisory Committee to the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration. Use of Challenge Studies in Evaluation of Cholera Vaccines. January 26, 1993, Bethesda, MD. 81. Haffkine WM. Les vaccinations antichole´riques aux Indes. Bull Inst Pasteur 1906; 4:697–737. 82. Haffkine WM. Protective inoculation against cholera. Calcutta 1913; 38–39. 83. Powell A. Further results of Haffkine’s anticholera inoculation. J Trop Med Hyg 1899; 2:115. 84. MacLeod CM, Hodges RG, Heidelberger M, et al. Prevention of pneumococcal pneumonia by immunization with specific capsular polysaccharides. J Exp Med 1945; 82:445–465. 85. Francis T Jr., Korns RF, Voight RB, et al. An evaluation of the 1954 poliomyelitis vaccine trials. Summary report. Am J Public Health 1955; 45(5 part 2):1–50. 86. Wilson GS. The Hazards of Immunization. London: University of London, Athlone Press, 1967. 87. Berlin Correspondent. The Lu¨beck trial. Lancet 1932; 1:259–260. 88. Berlin Correspondent. The Lu¨beck trial. Lancet 1932; 1:365. 89. Anonymous. The Vienna antitoxin controversy. Lancet 1926; 2:1074–1075. 90. Jakovleva JL. Polyneurite diphthe´rique consecutive a` l’injection de toxine diphthe´rique. Excerpted in: Bull Inst Pasteur 1927; 25:312. 91. Ten Broeck C, Chuan SH, Koch R. Diphtheria toxin-antitoxin mixture. China Med J 1927; 41:414–423.

Chapter 1: Vaccines and Vaccination in Historical Perspective 92. Annotation. The Bundaberg tragedy. Med J Aust 1928; 2:31–32. 93. Tigertt WD. The initial effort to immunize American soldier volunteers with typhoid vaccine. Mil Med 1959; 134:342. 94. Editorial. Jaundice following yellow fever vaccination. JAMA 1942; 73:1110. 95. Findlay GM, Martin NH. Jaundice following yellow fever immunization: Transmission by intranasal installation. Lancet 1943; 1:678. 96. Seef LB, Beebe GW, Hoofnagle JH, et al. A serologic follow-up of the 1942 epidemic of post-vaccination hepatitis in the United States Army. N Engl J Med 1987; 316:965–970. 97. Nathanson N, Langmuir AD. The Cutter incident. Poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the United States during the spring of 1955: I. Background. Am J Hyg 1963; 78:16–28.

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98. Nathanson N, Langmuir AD. The Cutter incident: poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the United States during the spring of 1955. II. Relationship of poliomyelitis to Cutter vaccine. Am J Hyg 1963; 78:29–60. 99. Nathanson N, Langmuir AD. The Cutter incident. Poliomyelitis following formaldehyde-inactivated poliovirus vaccination in the United States during the spring of 1955: III. Comparison of the clinical character of vaccinated and contact cases occurring after use of high rate lots of Cutter vaccine. Am J Hyg 1963; 78:61–81. 100. Langmuir AD, Bregman DJ, Kurland LT, et al. An epidemiologic and clinical evaluation of Guillain-Barre´ syndrome reported in association with the administration of swine influenza vaccines. Am J Epidemiol 1984; 119:841–889.

2 Developing Vaccines in the Era of Reverse Vaccinology Rino Rappuoli Novartis Vaccines and Diagnostics S.R.L, Siena, Italy

Gordon Dougan Microbial Pathogenesis, Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K.

Isabel Delany Molecular Genetics Unit, Novartis Vaccines and Diagnostics S.R.L., Siena, Italy

INTRODUCTION

Recombinant DNA Technology

Vaccines are currently available for infectious diseases caused by various viruses and bacteria, and the prevention of disease and death by vaccination has profoundly improved the public health of many populations globally. However, vaccines are not yet licensed for use against many other important infectious diseases, and new or improved vaccines are needed to replace suboptimal vaccines and to address newly emerging pathogens. New vaccines are being introduced at a higher rate than ever before. Half of all new vaccines have been developed in the past 25 years, at a rate of approximately one per year over that time frame, compared with an average of one every five years before that. For more than a century, vaccine development has followed Pasteur’s principles: ‘‘isolate, inactivate and inject’’ the causative microorganism (1). The majority of vaccines currently licensed and available for human use include live, attenuated organisms and killed or inactivated organisms. A small subset is based on partially purified components of an organism and even fewer still are recombinantly produced vaccines. However, as we come to address the problems of organisms for which no effective vaccines are currently available, it will become increasingly important to turn to novel approaches offered by advances in biotechnology. Recently, vaccines against cervical cancer have been licensed on the basis of human papillomavirus (HPV) virus-like particles (VLPs) produced in yeast or insect cells (2). The ongoing discovery and application of innovative technologies should continue to revolutionize the way vaccines will be made in the future, generating vaccines against even the most challenging pathogens.

Recombinant DNA technology was first successfully applied to vaccines over 25 years ago, with the production of a recombinant vaccine for Hepatitis B virus (HBV) (4). Although a vaccine based on purified HBV surface antigen (HBSAg), from the plasma of infected patients, was available since the 1970s, the expression in yeast of the HBSAg vaccine antigen provided the solution for a relatively simple, quick, and inexpensive process to produce a safer vaccine. This development also hallmarked the first VLP vaccine, as the recombinant HBSAg was able to self-assemble, facilitating purification and manufacture at the industrial level. Since this initial success, production of protein antigens using recombinant DNA techniques has become a standard practice for subunit vaccine development. Along with the need for developing better-characterized (e.g., HBV), less reactogenic (e.g., acellular pertussis), or more potent vaccines (e.g., anthrax) to replace the existing vaccines, a more recent driver for developing recombinant subunit proteins has been the need to provide broader protection against multiple strains or serotypes of a bacterium (e.g., serogroup B meningococcus, pneumococcus). Strategies to increase the breadth of vaccine coverage include the engineering of multiple epitope-based vaccines, incorporating epitopes either from different antigens of the same pathogen or even from different pathogens into a single protein using synthesized genes. Another major use of recombinant DNA technology has been in site-specific inactivation of toxins as a safe and efficient alternative to chemical inactivation. A successful example of this is the genetic inactivation of the pertussis toxin for incorporation into the acellular pertussis vaccine. Detailed structurefunction analysis of the pertussis toxin allowed the identification of key amino acids responsible for the toxicity of the protein that could be mutated, thereby inactivating the toxin while maintaining antigenic conformation (5). This safe and immunogenic protein along with two other purified components, filamentous hemagglutin and pertactin, make up the acellular pertussis combined subunit vaccine and replaced the traditional killed whole-cell vaccine (6). Similarly, the sitedirected mutation of toxins for use as the protein carrier in conjugated polysaccharide vaccines exemplifies an extrapolation of the diverse areas of vaccine design and development to

TECHNOLOGICAL REVOLUTIONS IN VACCINE RESEARCH The history of vaccine development can be marked by a number of milestones resulting from revolutions in technology. Earlier waves involved pathogen attenuation, inactivation, and viral cell culture, all supported by an increased understanding of the human immune response (3). However, recombinant DNA technology has probably been the single most innovative advance that has opened the door to new technological developments.

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which recombinant DNA technology has been applied. Ultimately, recombinant DNA technology is forming the basis of a wide range of new platforms for vaccine delivery and exploitation of expanding immunological knowledge, examples of which are discussed below.

Engineering Live Attenuated Vaccines The use of recombinant DNA has been applied to the genetic attenuation of bacteria and viruses for use in live attenuated vaccines. Previously, attenuation of bacteria or viruses was achieved through serial in vitro passage or random chemical mutagenesis, with the potential accumulation of unknown and uncharacterized mutations and the risk of reversion. By contrast, recombinant, attenuated vaccines are specifically engineered to inactivate defined target functions with nonreverting mutations. Furthermore, the present requirements for licensing new vaccines are more rigid than that in the past and call for strains that are well defined and carry precisely defined mutations. The rational design behind genetically engineering a recombinant vaccine strain should find the right balance between attenuation and immunogenicity. Initial attempts to generate recombinant, attenuated vaccines gave rise to unacceptable levels of reactogenicity in humans, and many promising candidate strains failed in clinical trials. To achieve balanced attenuation, different combinations of mutations can be introduced, which group into two main types, those that target critical housekeeping functions and those that target disease-related virulence factors. For example, auxotrophic mutations have been engineered by deleting genes in essential metabolic pathways. One of the most commonly used mutations for generating vaccines for intracellular pathogens such as Samonella and Shigella are aro mutations, which disable the shikimate pathway essential for the biosynthesis of aromatics including the aromatic amino acids. A second method is the targeting of critical virulence genes such as those encoding expression of the Salmonella pathogenicity island 2 (SPI2) type III secretion systems of Salmonella or the ctx toxin of Vibrio cholerae. Currently, candidate live vaccine strains are being constructed that combine different mutations, which optimize the balance between attenuation, reactogenicity, and immunogenicity, highlighting the need to understand the organisms’ physiology and interaction with the host as well as protective immune responses to the disease. Live attenuated virus vaccines are also being designed through reverse genetic approaches. For instance, attenuated dengue viruses have been generated by sequence modification or deletion and, alternatively, by producing recombinant antigenic chimeras between two related viruses (7). Recombinant DNA techniques could also facilitate the rapid generation of genetically attenuated viruses from emerging infections, such as metapneumovirus (8), or in response to the emergence of a new influenza variant. The use of reverse genetics enables rapid production of reference influenza vaccine viruses, and this has been exploited for the generation of an inactivated whole-virionbased vaccine for the influenza H5N1 reference vaccine strain in response to the latest pandemic flu threat (9,10).

Live Attenuated Bacteria as Vectors There is increased interest in the use of live attenuated bacterial vaccines (LBVs) as carriers for the presentation of heterologous antigens for the engineering of live, recombinant mucosal vaccines. LBVs allow vaccination through mucosal surfaces

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and specific targeting of professional antigen-presenting cells located at the inductive sites of the immune system (11). Both humoral and cellular immune responses can potentially be primed by this approach. Bacterial species that are being investigated as vector vaccines include attenuated strains of Salmonella enterica serovar typhi and serovar typhimurium, Shigella, V. cholerae, Listeria monocytogenes, bacille Calmette-Guerin (BCG) derived from Mycobacterium bovis, and Yersinia enterocolica. Other bacterial vectors have included nonpathogenic strains derived from the normal flora such as Streptococcus gordonii, Lactobacillus casei, and L. lactis (12). Salmonella strains are of particular interest since these strains can be administered orally and may induce mucosal as well as systemic immune responses. Furthermore, more than 20 years of experience with a licensed live attenuated Salmonella vaccine, S. typhi Ty21a (Vivotif1, Berna Biotech, a Crucell company, Berne, Switzerland) is available and indicates that this strain is safe and effective in vaccination against typhoid fever (13). The generation of new carrier strains as well as improved systems of in vivo expression and localization (e.g., surface vs. internal localization and/or targeting to different cell compartments) of heterologous proteins is the focus of many groups’ efforts (14,15). A further novel approach exploits intracellular bacteria as delivery vectors for DNA vaccines (11). Some bacteria have been shown to deliver DNA vaccines to human cells in vitro and have provided evidence for in vivo efficacy in several experimental animal models of infectious diseases and cancers.

Conjugate Vaccines The use of protein-conjugated polysaccharides as a tool for the prevention of diseases caused by encapsulated bacteria has proved to be highly successful in the development of effective vaccines against Haemophilus influenzae, Neisseria meningitidics, and Streptococcus pneumoniae. Subunit vaccines based on purified capsular polysaccharide can elicit protective immune responses in adults and are the basis of licensed multivalent pneumococcal and meningococcal polysaccharide vaccines. However, these have not been widely exploited as they were shown to result in short-lived protective immunity and to be ineffective in infants, largely because of their inability to engage a T cell–based immune response. The development of conjugate vaccines derived from polysaccharides of the capsule chemically conjugated to tetanus toxoid or diphtheria CRM197 carrier proteins resulted in a T-dependent antigen capable of protecting young children and providing long-term immunological memory. The recent success of the heptavalent conjugated vaccine (Prevenar) against pneumococcus, which protects against the seven serotypes that most commonly cause invasive pneumococcal disease in infants and young children, is an example of the development of an improved conjugate vaccine (16). To date, the polysaccharide used in large-scale vaccine production has been purified from the pathogen itself, grown in large quantities—an approach that is costly and difficult to control. The large-scale production of a conjugate vaccine containing synthetic polysaccharides has been recently achieved (17). Through simplification of the carbohydrate chemistry involved, the first large-scale production of a H. influenzae type b vaccine, consisting of synthetic polysaccharide conjugated to tetanus toxoid protein carrier, was demonstrated. This vaccine has been shown to be as efficient as

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commercially available nonsynthetic vaccines in inducing protective levels of antibodies in infants.

Virus-Like Particles VLPs are structures resembling a virus but empty of nucleic acids, which are derived from self-assembling subunits of virus structural antigens. VLP vaccines combine many of the advantages of whole-virus vaccines (induction of strong immune responses) and recombinant subunit vaccines (relative simplicity in manufacturing a safe vaccine). Commercialized VLPbased vaccines have been successful in protecting humans from HBV and HPV infections. These vaccines have excellent safety profiles, are highly effective, and induce long-lasting antibody responses. VLP vaccines are currently being explored for their potential to combat other infectious diseases and cancer. Many VLPs, including HPV VLPs, do not require coadministration of exogenous adjuvants to induce strong antibody responses. In addition, VLPs efficiently induce T-cell responses through interactions with antigen-presenting cells, particularly dendritic cells. VLPs are routinely expressed and produced from yeast, mammalian, and insect cells (the baculovirus expression system). VLPs designed for mucosal vaccination can also be efficiently produced in gut bacteria such as highly attenuated Salmonella or Lactobacillus strains and even in plants for oral inoculation (18). VLPs have also been exploited as molecular scaffolds for heterologous antigen presentation. Many different VLPs have been adapted for this purpose by incorporating heterologous epitopes into already well-characterized surface loops of virus antigens, generating chimeric particles. Since multiepitope vaccines have been shown to be more successful in inducing broad immune responses, a second generation of VLPs has been designed incorporating epitopes from more than one antigen of the same viral agent or antigens for different pathogens in a combined vaccine. A successful example of this is the synergistic effect resulting from vaccination with a combined CombiHIVvac vaccine, which incorporates B-cell and T-cell epitopes from Env and Gag proteins in a multicomponent VLP containing a DNA vaccine encoding multiple immunogens of HIV-1 (19). The level of antibodies induced by immunization with any of the immunogens was significantly lower compared with that induced by the combined vaccine. Lessons learned from combating well-known viruses like HBV, HPV, or HIV are constantly being carried over to newly emerging and less intensively studied viral diseases, for which VLP-based strategies might serve as attractive first-step tools to develop protective vaccines. Furthermore, the VLP-based technological platform is being exploited for innovative new vaccines directed against nontraditional targets such as selfantigens involved in chronic diseases (e.g., rheumatoid arthritis) or for vaccines toward a better standard of living such as the antismoking vaccine that targets nicotine. Through chemical conjugation of nicotine to a VLP-based vaccine, it was demonstrated that, when a sufficient antibody level is achieved, continuous abstinence rates can be significantly increased by vaccination (20).

Replicating and Nonreplicating Vectors Historically, live attenuated, replicating vaccines, such as measles, mumps, rubella, polio, vaccinia, and yellow fever, rather

than inactivated preparations, have provided the most effective protection against viral infection and disease. Notably, these vaccines elicit essentially lifelong protective immunity (21). The idea of using viruses as gene delivery vehicles to combat diseases has been an obvious next step. The failed efforts to develop effective vaccines against AIDS and malaria led to the development of a wide range of innovative viral vectors that are able to efficiently deliver antigens and induce immune responses (22). A broad spectrum of replicating and nonreplicating vectors are available. A variety of attenuated viruses have been employed as vectors including vaccinia and other pox viruses, adenovirus, and single-stranded RNA virus replicon vectors such as alphaviruses, coronaviruses, picornaviruses, flaviviruses, influenza viruses, rhabdoviruses, and paramyxoviruses. Choice of an appropriate vector for use in the development of a vaccine depends on the biology of the infectious agent targeted, as well as multiple other factors. These include whether the vaccine is intended to prevent infection or to boost immunity in already infected individuals, prior exposure of the target population to the vector, safety considerations, the number and size of gene inserts needed, and suitability for large-scale manufacturing and compliance with regulatory requirements. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Most vectored vaccines are designed to elicit cellular immune responses. In prime-boost regimens, vector priming followed by a booster inoculation with a protein antigen can induce broad and potent antibodies. Alternatively, some viral vectors such as alphavirus can be engineered to elicit potent antibody responses (23) and might prove to be a useful alternative (21). Use of recombinant replication-proficient and nonreplicating vectors will face extensive preclinical testing and will possibly have to meet stringent regulatory requirements. However, some of these vectors may benefit from the profound industrial and clinical experience of the parent vaccine (22). For instance, among the promising vectors is the measles virus, with a long-standing safety and efficacy record. The measles vaccine induces strong cellular and humoral immune responses after a single injection, and it is likely that a multivalent vaccine could be produced on a large scale and at low costs similar to the parental vaccine, making it an attractive model platform for the development of live attenuated, recombinant vaccines. However, even in this case, the modified recombinant vectors will need to be carefully tested for safety to make sure that the modification has not introduced unexpected properties.

DNA Vaccines The discovery in the early 1990s of DNA immunization radically changed accepted views of the nature of a vaccine. Instead of delivering an antigen per se, genetic material that encodes a specific antigen is delivered into mammalian cells, directing expression of the antigen by the cell itself, essentially working from the inside out. Numerous viral, bacterial, and parasitic antigens have been delivered by DNA vaccines, with varying success rates to date. The high expectations associated with DNA vaccination, as a result of promising data obtained in animals, were somewhat tempered by disappointing early results when DNA was tested as a vaccine in humans. A recognized limitation of DNA vaccines is their limited capacity to induce good virus-neutralizing antibody responses. New

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strategies have been developed to improve or amplify the immunogenicity of DNA vaccines. Studies conducted over the last few years have led to promising results, particularly when DNA is used in combination with other forms of vaccines (24). A particularly promising approach is a heterologous ‘‘prime-boost’’ strategy, where administration of plasmid DNA is followed by recombinant virus [modified vaccinia virus Ankara (MVA) or adenovirus] expressing the same antigen (chap. 38). However, it is increasingly apparent that the immunogenicity of DNA vaccines greatly depends on the delivery method used for immunization. Therefore, the latest generation of DNA vaccines may rely on improved delivery either through the use of microparticles or through viral vectors or bacterial vectors. The coadministration of genes encoding regulatory cytokines or other immunostimulatory cytokines is an attractive approach to improve or modify responses to DNA vaccines, although it brings with it an alternative set of safety concerns.

THE GENOMIC REVOLUTION The more recent genome revolution has extended the boundaries in vaccine research. Genome sequences offer unprecedented access to all the possible antigens of an organism and furthermore enable the possibility of rational selection of targets depending on the desired objective. Before genomics, the conventional approach to vaccine development was based on time-consuming dissection of the pathogen using biochemical, immunological, and microbiological methods. For pathogens that do not grow in vitro, the availability of the genome sequence has enabled the development of recombinant vaccines, as has been carried out for HBV and is under way for hepatitis C. Since the first bacterial genome sequence was completed, that of H. influenzae (25), there have been enormous advances in both sequencing strategies and bioinformatic functional assignments, resulting in an exponential growth of sequence information. Complete genome sequences are available for 978 bacteria (as of August 2009), including at least one for all the major human pathogens and often multiple strains of the same species, with a further 1019 draft assemblies available, and the total ongoing number is closer to 3100 (26,27). The study of these genomes by both computational and experimental approaches has significantly advanced understanding of the physiology and pathogenicity of many microbes and provided insights into the mechanisms of genome evolution as well as microbial population structure (28). Genome mining and comparative genomics are allowing us to increase the number of candidate vaccine antigens by several orders of magnitude and make it possible to select antigens that are conserved or specific to pathogenic organisms (Fig. 1). The ability to globally view the expression of potential antigens on a genome-wide scale with innovative functional genomics technologies is also significantly changing the field of antigen discovery and vaccine design (Fig. 2). Our capacity to generate and analyze genome sequences has grown at an astonishing rate, but we are on the cusp of yet another leap forward, with the advent of a new family of ultrahigh-throughput, low-cost sequencing methods (29). These new sequencing strategies with combinations of capillary sequencing (30) and pyrosequencing (31) mean that, incredibly, the time necessary to complete the sequencing of an entire bacterial genome is now less than a day. New sequence assembly protocols and

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improved functional assignment methods mean that the bottleneck of closing genomes and annotation is no longer a difficult, tedious hurdle. However, growth of sequence data has brought with it new challenges. There is an ever-increasing need to develop tools and resources that can facilitate the understanding of the biological function of the thousands of uncharacterized gene products that have been identified through sequencing. The large proportion of genes that still need a functional assignment warrant a concerted effort in future research.

Reverse Vaccinology Genome mining has revolutionized the approach to vaccine development and provided a new innovation to antigen selection and design. The approach starting from the genomic information leading to the identification of potential vaccine candidates is termed ‘‘reverse vaccinology’’ (32). The availability of complete bacterial genome sequences offers a comprehensive catalog of genes encoding all the potential protein antigens of a pathogen, with the possibility of rational selection of vaccine candidates rather than empirical testing of antigens, one at a time. Furthermore, the prediction of antigens is independent of the need to culture the pathogen in vitro, to establish the abundance of the antigen, and to understand which antigens will be expressed in vivo. On the basis of the concept that surface-exposed antigens are susceptible to antibody recognition and are therefore the most suitable vaccine candidates, a complete genome can be screened using bioinformatic algorithms to select open reading frames (ORFs) encoding putative surface-exposed or secreted proteins. Surface proteins are readily identified in genomic data based on the combination of several pieces of information, including the presence of amino acid motifs that are responsible for targeting the mature protein to the outer membrane (signal peptides), to the lipid bilayer (lipoproteins), to the integral membrane (hydrophobic transmembrane domains), or by searching for motifs indicative of surface location such as sortase attachment sites (otherwise known as LPXTG sites) or motifs for recognition and interaction with host proteins or structures (e.g., adhesins having integrin-binding domains). Furthermore, screening for sequences homologous to those of human proteins for their exclusion in the selection process can help to avoid problems of autoimmunity. Currently available software and bioinformatic techniques for antigen discovery have been recently reviewed (33). Furthermore, current efforts are aimed at refining algorithms to enable biotechnologists to follow ‘‘computer-aided strategies’’ based on experiments driven by high-confidence predictions. Some groups are currently involved in developing and tuning integrative approaches and user-friendly, automated bioinformatics environments. For example, NERVE (New Enhanced Reverse Vaccinology Environment) (34) represents a user-friendly software environment, which attempts to bring together a similar body of principles into an automated pipeline resembling a reverse vaccinology process. After candidate surface antigens are identified in silico, they are produced in vitro and their immunogenicity is assayed to measure their relative power as potential vaccine candidates. The first application of this genomic approach led to the identification of novel vaccine candidates against N. meningitidis serogroup B (MenB).

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Figure 1 Developing vaccines through genomics: a new interdisciplinary realm of inquiry. The advent of genomics has revolutionized the way in which potential vaccine candidates against bacterial pathogens are identified. The genome sequences represent an inclusive virtual catalog of all the potential vaccine candidates, which may be mined to select the molecules that are likely to be effective through either ‘‘reverse vaccinology’’ or ‘‘epitope fishing’’ approaches. Genome comparisons of strains representative of genetic diversity can be a powerful tool for selection of broadly protective combinations of proteins as well as the identification of virulence factors, which may be exploited for attenuation. Functional genomics approaches are complementary to in silico antigen discovery and can contribute important information on in vivo expression, localization, and immunogenicity of antigens of an organism as well as functional roles of these proteins.

Pioneering Work of MenB: from One Genome to Universal Vaccine MenB is an example of where several decades of conventional vaccine development had been unsuccessful because the components identified by conventional approaches were identical to self-antigens or were hypervariable in sequence. In collaboration between Chiron Spa and The Institute of Genomic Research (TIGR) (35), the complete genome of the virulent strain MC58 was sequenced (36), and within 18 months of beginning the sequencing, more than 600 potential vaccine candidates had been predicted using computer analysis; 350 of these were expressed in Escherichia coli, purified and tested for their ability to elicit protective immunity (37). Antisera raised against the purified proteins were assayed for specificity (by Western blot), accessibility on the surface of the pathogen (by flow cytometry), and their ability to kill bacteria when combined in vitro with human complement (bactericidal assay). The ability to evoke bactericidal antibodies, inducing

complement-mediated killing of the bacteria, correlates with an antigen’s capability of conferring protection against the organism (38). This approach identified 28 novel protective antigens, several of which were conserved in a panel of strains representative of the meningococcal population and therefore likely to induce immunity against all meningococcal isolates. In essence, in under a year and a half, reverse vaccinology applied to MenB enabled the identification of more vaccine candidates than had been discovered during the previous 40 years by conventional methods. Moreover, the antigens identified using the genomebased strategy were different from those identified using conventional approaches. To strengthen the protective activity of the single protein antigens and to increase strain coverage and to avoid escape mutants, the final vaccine formulation comprises a ‘‘cocktail’’ of the selected antigens. These promising vaccine candidates are currently being tested in clinical trials (39) and are giving promising results. Importantly, this work also shows that

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Epitope Predictions and Immunomics

Figure 2 The pan-genome is the global gene repertoire of a bacterial species and depends on analysis of the number of available genome sequences. The size of the pan-genomes of Streptococcus agalactiae (GBS) and Bacillus anthracis are shown as a function of the number of sequenced strains. The curves represent a mathematical extrapolation of the data to a large number of strains. The pan-genome is made up of the core genome (the pool of genes shared by all the strains of the same bacterial species) and the dispensable genome (the pool of genes present in some, but not all, strains of the same bacterial species). Analysis of eight GBS genome sequences indicates that the pan-genome contains 2713 genes, of which 1806 belong to the core genome and 907 to the dispensable genome (56). Either the size of a species pan-genome can grow with the number of sequenced strains and is nominated an ‘‘open’’ pan-genome (the GBS pangenome), or the size can quickly saturate to a limiting value and is nominated a ‘‘closed’’ pan-genome (the B. anthracis pan-genome). GBS pan-genome is predicted to grow by an average of 33 new genes every time a new strain is sequenced (56). The final number of genes in the pan-genome of a species with an open pan-genome may be several orders of magnitude larger than the number of genes in an individual genome. Abbreviation: GBS, group B Streptococcus.

universal protein-based vaccines can be developed against encapsulated bacteria that are usually targeted by conjugate vaccines. In addition, one further advantage of reverse vaccinology is that all the antigens are produced as soluble recombinant proteins in E. coli, thus supporting the straightforward development of a suitable manufacturing process for largescale production. After the initial success of reverse vaccinology in solving the problem of antigen selection for MenB, reverse vaccinology has been applied to the search for antigens and virulence factors in many other pathogenic bacteria including Bacillus anthracis (40,41), Porphyromonas gingivalis (42), Streptococcus pneumoniae (43), Streptococcus agalaticae (44), Chlamydia pneumoniae (45), and Brucella melitensis (46). Thus, the reverse vaccinology approach appears to be applicable to a range of pathogens and, in principle, also to eukaryotic parasites, for which genome sequence and suitable in vitro or in vivo models are available.

For a vaccine to be effective, it must invoke a strong response from both T cells and B cells; therefore, epitope mapping is a central issue in vaccine design (47,48). In the past, scientists isolated proteins from whole cells and then digested the protein antigens to find smaller fragments or epitopes that stimulated the T-cell and B-cell response. The set of pathogen epitopes that interface with the host immune system is now known as the ‘‘immunome.’’ A key focus of immunomics has been the development of algorithms for the design and discovery of new vaccines. T-cell epitope-mapping algorithms are based on straightforward mathematical analyses of the patterns of amino acids that occur in peptides bound to (and presented in the context of) human leukocyte antigen (HLA) by antigenpresenting cells (47). Because the epitope peptide is bound in a linear form to HLA, the interface between ligand and T cell can be modeled with breathtaking accuracy. In contrast, B-cell epitope-mapping algorithms have lagged behind T-cell epitope-mapping algorithms, and few B-cell epitope-mapping algorithms are in current use. In a whole-genome approach, similar to reverse vaccinology, a pathogen’s entire proteome in silico can be analyzed using T-cell epitope-mapping tools and further in vitro evaluation to discover new protein candidates for vaccines. The concept has been described as fishing for antigens using epitopes as bait. A number of recent papers point a clear path from the genome to the immunome, bringing us closer to understanding just what information about a pathogen is required for effective host immune defense (48–50). Largescale screening of pathogens for T- and B-cell epitopes from the National Institute of Allergy and Infectious Diseases (NIAID) categories A to C pathogens and other pathogens of global importance is also currently under way (51). This study includes a database of the complete systematic genome mapping of pathogens such as B. anthracis, M. tuberculosis, Clostridium tetani, Francisella tularensis, Y. pestis, and viruses including smallpox, flaviviruses, arenaviruses, rabies, influenza, and hepatitis A. Immunomics results in the expansion of a number of different proteins that can be screened for vaccine development while narrowing this search to regions of the proteins that are likely to induce an immune response (52). Researchers are now implementing these combined methods to scan genomic sequences for vaccine components. There are however some limitations to the ‘‘genome-tovaccine’’ approach. One obvious point of contention is the choice of the genome/genomes to mine. While often the choice of which genome to mine was essentially the first genome that was sequenced, this may not always represent an adequate representative organism. Today multiple genomes from each species are increasingly becoming available, opening the era of the pan-genomic reverse vaccinology, which will be dealt with further below. Another limitation of genome-derived vaccines is the inability to identify nonprotein antigens including polysaccharides or CD1-restricted antigens such as glycolipids, or the inability to identify posttranslational modifications on the selected protein antigens. Furthermore, there is a lack of algorithms that can be used to make a good correlation between antigens and their likely efficacy in protection especially for humoral immune responses (neutralizing antibodies), although such tools are now being developed. In addition, development of effective vaccines through genome mining is dependent on the available methods to measure in vitro efficacy and correlates of protection and not simply on identifying epitopes that

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are immunogenic. Therefore, the vaccine antigens identified must be evaluated in appropriate challenge models, and limited availability of good animal models may slow the progress to vaccine trials.

COMPARATIVE GENOMICS The availability of genome sequence information now allows us to compare species and strains within species for the identification of conserved genes and putative virulence factors. In particular, the analysis of the genetic variability between pathogens and closely related nonpathogenic microorganisms leads to the rapid identification of the complete set of genes potentially responsible for acquisition of virulence. This offers a valuable guideline into the search for suitable proteins to use as purified antigens in subunit-based vaccines. Vaccines should ultimately target antigens that are conserved among pathogenic strains. Therefore, comparative genomics can be used to find antigens that are likely to confer broad protection. This approach can also provide the rational basis for a safe and stable attenuation of live vaccine candidates or vectors for vaccine delivery. Comparisons can be performed either with genome sequence or by using microarray-based methods. Owing to the improvement of sequencing technologies and the consequent reduction of sequencing costs, multiple genome sequences have been completed for several species over the past few years that enable quantitative analyses of their genomic diversity through comparative genomic analyses. Some pathogens exhibit very little, while others have marked genetic variability. For example, M. tuberculosis is now recognized to be an intracellular clonal bacterium that harbors relatively little genetic diversity. Studies based on whole-genome comparisons use single-nucleotide polymorphisms (SNPs) to investigate M. tuberculosis evolution and phylogeny (53). However, there is increasing evidence that the interstrain variation that exists is biologically significant; for instance, underlying biological differences among clinical strains have been associated with an adaptation to a specific host range or a response to variations in vaccination practices. On the other hand, E. coli represents a very wide group of organisms that have high levels of intraspecies diversity that vary in as much as 25% of their genome (54). E. coli are part of the natural gut flora as commensals but can also cause diverse infections in very different niches. Genomic variations that occur in the form of individual genes or larger genome islands contribute to differences in virulence potential. Isolates of the same species can be analyzed experimentally by subtractive hybridization and comparative genome hybridization (CGH). Microarrays spotted with predicted ORFs of a reference strain can be hybridized with labeled DNA from an experimental strain, allowing genes common to both, as well as those present in the reference strain but absent in the test strain, to be identified. CGH allows high-throughput, high-resolution global genome analysis without the need to sequence all strains tested. In a recent study, microarrays based on the genome sequence of CFT073 were utilized in CGH analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. This approach resulted in the identification of 131 genes that were exclusively found in ueopathogenic E. coli (UPEC) relative to commensal and fecal isolates (55). However, this highlights one intrinsic technical limitation of microarray: detection is limited to the DNA spotted on the array.

The Pan-Genome Multiple genomes of the same species and comparative genomics have led to an increased understanding of the intraspecies diversity, making clear that the sequence of one genome may not be sufficient to represent the genetic diversity of a microorganism. To overcome this limitation, the pan-genome concept was introduced (56) to define the global gene repertoire possibly pertaining to a given species. The unexpectedly high degree of intraspecies diversity suggests that a single genome sequence is not representative of the genetic inventory of a given taxonomic group but is rather a sampling of genes characterizing members of a given population in the same gene pool. In the seminal work of Tettelin et al., authors set about answering the question of how many genomes are needed to fully describe a bacterial species using eight genomes representative of the diversity among group B Streptococcus (GBS) (56). Comparative analysis of the genomes enabled the estimation that 1806 genes are shared by all strains of Streptococcus agalactiae, and these genes form the species ‘‘core genome.’’ This represents approximately 80% of the average number of genes encoded in each strain and, in general, includes all genes responsible for the basic aspects of the biology of a given species. Instead, the ‘‘dispensable genome’’ is composed of genes absent or partially shared and strainspecific genes, and these genes are responsible for species diversity and might encode functions that can confer selective advantages. Surprisingly, mathematical extrapolation of the existing data predicted that, no matter how many strains have been sequenced, each new sequence would contain genes that have not been encountered before, leading to the counterintuitive conclusion that this species pan-genome continues to grow without bounds as the number of sequenced strains grows (Fig. 2), defined as an ‘‘open’’ pan-genome. It was estimated that the sampling of subsequent genomes would continue to reveal new genes, on average 33 per genome (56). However, the extent of intraspecies diversity is not always so vast, and a different behavior was observed in the study of eight independent B. anthracis isolates. In this case, the number of specific genes added to the pan-genome was found to rapidly converge to zero after the addition of only a fourth genome (56) (Fig. 2). Hence, the B. anthracis species has a ‘‘closed’’ pan-genome, and four genome sequences are sufficient to completely characterize this species, at least in terms of gene content (Fig. 2). A subsequent analysis of seven E. coli genomes has shown an extreme flexibility, with each new sequenced strain contributing 441 new genes to the core genome comprising 2865 genes, leading again to an open pangenome (57). Mathematical modeling suggests that hundreds of genomes of other species will follow the same trend (58). Given that the number of unique genes is vast, the pan-genome of a bacterial species might be orders of magnitude larger than any single genome (58). In conclusion, species can have an open or a closed pangenome. An open pan-genome is typical of species that either colonize multiple niches or have efficient mechanisms, such as natural competence, of exchanging genetic material with unrelated species present within the same environment (e.g., Helicobacter pylori, E. coli, N. meningitidis, and Streptococcus). By contrast, other more clonal species (such as B. anthracis, M. tuberculosis, and C. pneumoniae), which are more conserved, live in isolated niches with limited access to the global microbial gene pool and, therefore, have a low capacity to acquire foreign genes. Data from multiple

Chapter 2: Developing Vaccines in the Era of Reverse Vaccinology

genomes is necessary for a complete understanding of pathogen biology, as well as to understand better the diversity of bacterial species.

Lessons Learned While Exploiting the Pan-Genome of GBS The determination of a species pan-genome also allows us to infer important practical information for vaccine design. Although the core genes represent the most desirable source for the selection of conserved and therefore potentially universally applicable vaccine candidates, they are also more likely to be immunologically silent in any successful pathogen. The group of dispensable genes, by contrast, might be an invaluable source of novel antigens that, although only present in a subgroup of strains, might encode important virulence-associated functions and might be exploited in appropriate combinations to elicit a broad immune response. In particular, the pan-genomic concept in GBS was exploited for the discovery of an effective vaccine for cross-strain reactivity. A multigenome reverse vaccinology approach was undertaken (44), in which the core and dispensable genomes of the GBS pan-genome, resulting from analysis of the eight representative genome sequences, were mined for vaccine candidates. Among the predicted surface-exposed proteins, 396 were core genes and 193 were variable genes. The subsequent ability of the in vitro expressed candidates to confer protection in in vivo screening against a large panel of isolates resulted in the identification of a candidate universal GBS vaccine. This consists of a combination vaccine of four proteins, which is able to cover a wide range of strains. The important novelty of this study is that none of these antigens could be classified as universal, because only one of them is from the core genome (showing negligible surface accessibility in some strains), and the other three are encoded by dispensable genes and were therefore absent in a fraction of the tested strains. Therefore, the analysis of multiple genomes of GBS revealed tremendous diversity and identified candidates that are not shared by all the strains sequenced but provide general protection when combined. Intriguingly, it was found that all three dispensable proteins are components of pilus-like structures with an important role in the virulence in GBS (59). These structures, over four times the length of the bacterium itself, had remained completely elusive to researchers after decades of work in the field. Therefore, up-front genome comparisons from strains representative of the genetic diversity is a powerful tool for the selection of broadly protective combination of proteins and may be instrumental in formulation in universal vaccines against pathogens with highly diverse circulating strains. The natural next step to achieve a more comprehensive and epidemiologically related picture of bacterial populations will be population vaccinology, leading to the formulation of vaccines from a collection of proteins that, together, protect against the major circulating populations of a pathogen (60). To accomplish this, a more comprehensive and in-depth understanding of population structure is required. For many years it has become increasingly apparent that biology is no longer a linear science, but with the recent explosion in information due to metagenomics, we are beginning to understand we are at the tip of the iceberg. Understanding the variation that exists within populations seems miniscule when we think of the vast variation that is emerging through metagenomic sampling of global microbial

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populations. Over six million new proteins were predicted as the fruits of a single metagenomics project sampling global sea water (61), nearly doubling the total number of proteins known to date.

FUNCTIONAL GENOMICS ‘‘Postgenomic’’ methods (e.g., genomic microarray-based methods and proteomics) have changed the way investigators approach the classical questions: scientists now address questions on a whole-cell or system-wide basis, in contrast to the classical reductionist approaches. Genomics empowers the use of highly parallel methodologies that allow investigators to study all the genes or all the proteins of a pathogen in the context of a host or under various physiological or genetic states of interest (28). Functional genomics approaches are complementary to in silico antigen discovery. These include the large-scale analysis of gene transcription by DNA microarrays, the identification of the whole set of proteins encoded by an organism (proteomics) by two-dimensional gel electrophoresis and mass spectrometry, as well as using these protein reagents to create protein chip technologies to monitor immunological responses in human sera. Furthermore, the highthroughput capacity of these techniques facilitates the quantification of expressed genes in a comprehensive genome-wide framework. Before genomics, in vivo expression technologies (IVETs) (62) and signature-tagged mutagenesis (STM) (63) used promoter-trapping methods or inhibition of gene function to analyze genes highly expressed in vivo or important for infection. Needless to say, both technologies have greatly benefited from the availability of genome sequences, although the previous knowledge of the genome sequence is not strictly necessary for their application. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, highthroughput functional screens can now be achieved on a genomic scale (64).

Global Genomic Profiling Using Microarrays Global genomic profiling of gene expression using ordered DNA or oligonucleotide microarrays is a very powerful technology, which can be exploited in many different ways to further the study of genes that are involved in microbial pathogenesis. For vaccine discovery programs, it is of key importance to know what genes are expressed during host infection and what proteins can elicit an antibody response in humans. Microarray-based expression studies provide a strong contribution to the understanding of how a pathogen orchestrates responses to the host environment. DNA microarrays can be used to obtain a global profile of genes of a pathogenic microorganism whose expression is upregulated during infection of animals or of in vitro models. The major challenges in performing expression analysis in vivo or in vitro include the efficient recovery of RNA and the choice of an appropriate model system and/or experimental systems. The transcriptional changes in N. meningitidis were investigated from meningococci incubated in human serum as well as adherent to human epithelial and endothelial cells (65). The authors of this study discovered a wide range of surface proteins that are induced under in vivo conditions and that could represent novel candidates for a protein-based vaccine for meningococcal disease.

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Researchers are also using microarray technology to identify genes differently expressed in response to alteration in environmental parameters and to evaluate mutations or key factors in regulatory and metabolic pathways (66). This can aid in the functional characterization of protein antigens with as yet unidentified roles. Gene expression can be analyzed in either pathogen or host, thus allowing investigation of both sides of the hostpathogen interaction. Understanding the mechanism of protection of a vaccine is important for developing a new generation of vaccines. Some more recent studies have been directed toward obtaining immunological profiles of gene expression responses in individuals/in vitro models through DNA microarray following vaccination to assess the correlation of these parameters with protection. Global gene expression profiling is an ideal platform to compare induction of immune-response parameters following vaccination and challenge. For instance, human immunological responses to the F. tularensis live vaccine were monitored with transcriptome analysis (67) to gain a better understanding of the mechanism of protection afforded by the vaccine and perhaps the responses necessary for longlasting immunity. Many similar studies have been involved in the emerging field of ‘‘vaccinomics,’’ which encompasses immunogenetics and immunogenomics as applied to understanding the mechanisms of heterogeneity in immune responses to vaccines (68). This growing area of inquiry and importance seeks to understand the influence of immune-response gene polymorphisms on the heterogeneity of humoral, cell-mediated, and even innate immune responses to vaccines at both the individual and population levels.

is invaluable for identification of in vivo immunogens suitable as vaccine candidates (70–72).

Protein Array Technology Many groups are now involved in using the available genome sequence of an organism to construct a comprehensive gene collection for expression of the entire proteome of the organism. The proteins can then be spotted onto arrays, and these tools allow comprehensive analyses of immune responses and system-wide functional studies. LaBaer and colleagues have developed full proteomic arrays for the pathogens F. tularensis (73) and V. cholerae (74). Proteins expressed by pathogenic organisms can be screened with serum from convalescent patients to identify immunodominant antigens, leading to good vaccine candidates. A second application of protein microarrays is in examining protein function in high throughput by assessing their interactions and biochemical activities (75). JPT Peptide Technologies in Berlin, Germany, is examining similar applications, with microarrays displaying overlapping peptides comprising the full M. tuberculosis proteome. The analysis of reactivity profiles provides a wealth of novel information about the immune response against microbial organisms that would pass unnoticed in analysis of reactivity to antigens individually. Extension of this approach to a genome-wide fraction of the proteome may expedite the identification of correlates of protection and vaccine development against microbial diseases. In summary, coupled with new technologies such as protein microarrays, proteomics might allow the functional characterization of and the documentation of immune responses to each protein of a pathogen. These results in turn may lead to better understanding of the pathogen biology and new vaccine and therapeutic strategies.

Proteomics Recent years have seen the accelerated development of technologies that study proteins in high throughput. Referred to as functional proteomics, these methods support the global study of protein interactions, enzymatic activities, and immune responses. The complete complement of proteins of an organism following separation by two-dimensional protein separation methods can be analyzed/identified by mass spectrometric analyses. These analyses can be from both a qualitative and a quantitative point of view. Global protein expression profiles from two different conditions can be generated and compared using proteomics to identify up- or downregulated proteins. Proteomics can define proteins that are differentially located or secreted to outside of the cell (i.e., to the media or host cell). Rodrı´guez-Ortega et al. (69) described a new procedure using proteolytic enzymes to ‘‘shave’’ the group A streptococcal (GAS) surface, and the peptides generated were then separated and identified. This approach provided an extensive map of the surface antigens, namely, the ‘‘surfome’’ of the GAS strain, and enabled the identification of a new possible vaccine target. Use of this technique can provide a detailed picture of surface protein organization in any pathogenic bacterium. The combination of proteomics with serological analysis has led to the development of a new approach termed serological proteome analysis. After a two-dimensional separation of a pathogen’s protein sample, sera from individuals known to have been infected is used to identify immunoreactive proteins against which the patient has mounted a response. This method

STRUCTURAL PREDICTION AND STRUCTURAL GENOMICS After the storm of the ‘‘omics’’ era, there has been an increased understanding that it is also necessary to return to characterization of the individual proteins, putting a ‘‘face’’ on the molecules themselves. An increased effort to elucidate the structures of surface molecules is under way, and ongoing technological advances in protein biochemistry have allowed high-throughput platforms for structural resolution (76). Current structural genomics projects are being driven by two main goals: (i) to produce a representative set of protein folds that could be used as templates for comparative modeling purposes and (ii) to provide insight into the function of currently unannotated protein sequences. In target selection, a strong emphasis has been put to disease- and drug-related proteins. The number of high-resolution structures available in public databases today is approaching 50,000, including almost 30,000 protein structures, which will definitely aid in structure-based vaccine and drug design. The ultimate goal is to have at least one structure, and possibly multiple structures, from members of each protein family described to allow more accurate modeling. We believe that a systematic approach to the structural properties of immunodominant and immunosilent epitopes can provide the scientific rationale that in future may allow us to engineer immunodominant epitopes. A rational approach to the three-dimensional structure of antigens (structural vaccinology) is one of the basic aspects of vaccine research that should be a priority (77).

Chapter 2: Developing Vaccines in the Era of Reverse Vaccinology

SCORING VACCINES FOR PROBABILITY OF SUCCESS A recent review of history of vaccination concluded that the probability of success in vaccine development is highest when protection is mediated by antibodies and antigens that have no or limited antigenic variability (1). In Figure 3, we have tried to rank the panoply of vaccines discussed in this book according to two criteria, as variables: (i) the type of immunity that is critical for protection and (ii) antigenic stability. Vaccines that

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have the highest probability of technical success are in the upper right quadrant, which belongs to the pathogens that can be addressed by antigens that induce an antibody-mediated protection and that are not highly variable. The risk in vaccine development increases as one moves toward the intersection of the two axes (Fig. 3). Accordingly, vaccines where T-cell immunity is critical for protection or where protective antigens are highly variable have an increased likelihood of failure during development. It is clear from Figure 3 that the majority of the successful

Figure 3 The new generation of vaccines discussed in the chapters of this book are plotted as a function of the type of immunity required to confer protection against the disease on the y-axis and the stability/variability of surface antigens of the pathogen on the x-axis. The probability of success of a vaccine during development is highest when protection is mediated by antibodies and when antigens have no or only limited antigenic variability (1). The upper right quadrant represents vaccines with a low risk of failure during the development process. Moving down and left, we move through zones of medium and high risk of failure, with more difficult challenges when antigens are extremely variable and/or protection relies only on T cell–mediated immunity.

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vaccines currently available reside in the upper right quadrant and have been developed using well-established technologies. Modern preventive vaccines required for more difficult infectious organisms can build on expanding new technologies and immunological knowledge. Good examples are the several recombinant and conjugate vaccines already licensed, and several vaccines developed using genomics (reverse vaccinology) that are in development. Reverse vaccinology represents a revolution in vaccinology and a milestone in biotechnology. It illustrates how a complex biological problem such as vaccine design can be solved by addressing and integrating in silico predictions and high-throughput in vitro experimental analysis for the identification of optimal vaccine antigen formulations. Beyond the pioneering work with MenB, we see the development and maturation of the technique, as it has been adapted to other biological systems with their inherent difficulties (such as GBS). We also see how newly emerging bioinformatic and immunoinformatic technologies can be integrated with functional genomics approaches to hone down the targets (Fig. 1). With many more antigens discovered for each pathogen, it is now possible to select antigens that respond to validated principles, such as antibody-mediated protection, and with limited or absent antigenic variability. An important minority of the vaccines described in this book reside in the middle or left quadrant of Figure 3. These are the vaccines for which today’s technologies have not yet succeeded, and developing these vaccines requires bridging scientific gaps such as learning to develop vaccines based on T cell– mediated protection. Today, development of these vaccines is being addressed using innovative immunostimulatory molecules and adjuvants, replicating or nonreplicating viral vectors, prime-boost regimens, etc. An alternative approach could be to bring them to the ‘‘comfort zone’’ of the upper right quadrant of Figure 3 by learning how to engineer immunologically silent conserved epitopes into immunodominant epitopes. A good example of this is HIV, where antibodies such as B12 that recognize the conserved CD4-binding site could be able to protect from infection if this epitope were immunodominant. In conclusion, the majority of the new vaccines addressed in this book are within the reach of today’s technologies. Rather, the question is therefore whether or when they will be developed. Unfortunately, technical feasibility is only one of the hurdles in vaccine development. Even more important is often whether there is a market that can justify the huge investment that is necessary to bring vaccines to licensure. Many of the vaccines described in this book are at the ‘‘discovery’’ stage. However, the clinical development phase, during which a discovery is transformed into a potential product, is a long and expensive process. New vaccines today are developed by a few global vaccine manufacturers that can only afford to invest in vaccines that have a high probability of success in the market. In addition, the half dozen largest vaccine manufacturers have the traditional knowledge and the necessary investment to carry out clinical vaccine testing and manufacturing process development of candidate vaccine products. Many of the failures in vaccine development are due to the poor understanding and underestimation of the complexity of this phase. Thanks to the impressive progress in biotechnology, the vaccine field is embarking on a new post–genomic era: harnessing genome data, and using genomics, proteomics, and immunology techniques in a new interdisciplinary realm of inquiry (Fig. 1). Vaccines targeting hypertension, drug addiction, and

cigarette smoking are examples of how vaccines are being evaluated to target diseases that are not traditionally tackled by vaccination. Vaccines today are usually given to prevent diseases that parents and pediatricians have never seen, and in their minds, they are no longer immediate lifesavers but tools that improve the quality and duration of life.

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69. Rodrı´guez-Ortega MJ, Norais N, Bensi G, et al. Characterization and identification of vaccine candidate proteins through analysis of the group a Streptococcus surface proteome. Nat Biotechnol 2006; 24(2):191–197. 70. Klade CS. Proteomics approaches towards antigen discovery and vaccine development. Curr Opin Mol Ther 2002; 4(3):216–223. 71. Vytvytska O, Nagy E, Blu¨ggel M, et al. Identification of vaccine candidate antigens of Staphylococcus aureus by serological proteome analysis. Proteomics 2002; 2(5):580–590. 72. Ling E, Feldman G, Portnoi M, et al. Glycolytic enzymes associated with the cell surface of Streptococcus pneumoniae are antigenic in humans and elicit protective immune responses in the mouse. Clin Exp Immunol 2004; 138(2):290–298.

73. Murthy T, Rolfs A, Hu Y, et al. A full-genomic sequence-verified protein-coding gene collection for Francisella tularensis. PLoS ONE 2007; 2:e577. 74. Rolfs A, Montor WR, Yoon SS, et al. Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae. Proc Natl Acad Sci U S A 2008; 105(11):4364–4369. 75. LaBaer J, Ramachandran N. Protein microarrays as tools for functional proteomics. Curr Opin Chem Biol 2005; 9(1):14–19. 76. Lundstrom K. Structural genomics and drug discovery. J Cell Mol Med 2007; 11(2):224–238. 77. Serruto D, Rappuoli R. Post-genomic vaccine development. FEBS Lett 2006; 580(12):2985–2992.

3 Initial Clinical Evaluation of New Vaccine Candidates Carol O. Tacket and Karen L. Kotloff Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION Among the tools available to control infectious diseases, vaccines rank high in effectiveness and economic feasibility. Vaccines once consisted of either live whole virus analogues or killed virus or bacterial preparations. Now, new viral, bacterial, and parasitic vaccines are frequently defined by gene sequences or amino acid epitopes. Likewise, vaccine testing has progressed to become a discipline of its own, which includes scientific, epidemiologic, ethical, economic, and feasibility aspects. This chapter deals with some of these issues related to phase I and II vaccine testing.

SELECTION OF VACCINE CANDIDATES Creative and innovative vaccine candidates emerge from research laboratories in academic institutions, government agencies, and private pharmaceutical and biotechnology companies all over the world. The decision to begin human testing of a candidate vaccine depends on a number of criteria. First, the vaccine candidate must address a public health need and be a logical means of control for the disease of interest. For example, in the United States, outbreaks of Cryptosporidium parvum may best be prevented by improved water treatment rather than by vaccination. Similarly, infections with Shiga toxin producing Escherichia coli can best be prevented by improved meat inspection and consumer education about cooking practices, rather than by mass vaccination of children. Second, the vaccine candidate must have been designed with a sound scientific rationale. There are two mirror-image principles commonly used to develop vaccines. On the one hand, the vaccine may consist of a known or suspected protective antigen, for example, purified hepatitis B surface antigen or Haemophilus influenzae type b polysaccharide. Alternatively, the vaccine may be a live strain of a pathogen, attenuated by genetic deletion of known virulence factors, for example, live oral cholera vaccine CVD 103-HgR. Third, there must be an expectation of safety. The riskbenefit ratio for vaccines against most infectious diseases must be very low since such vaccines are designed for use in healthy individuals who may be at low risk of disease. In contrast, in the development of therapeutic agents, a larger risk may be acceptable since there is the opportunity for therapeutic benefit. Safety of the vaccine candidate, therefore, must be formally demonstrated in an appropriate animal model using a dose and route of administration that is proposed for clinical studies. Fourth, there must be animal studies demonstrating the immunogenicity of the product when given in the appropriate

dose and by the appropriate route and, if possible, a demonstration of efficacy against challenge with the wild-type pathogen in animals. Animal models to demonstrate immunogenicity and efficacy against challenge have been developed for a number of vaccines, for example, cotton rats for respiratory syncytial virus and mice for Salmonella. Fifth, it is desirable that the vaccine be prepared in a practical formulation at the onset of phase I studies. This is not an absolute requirement since it is often necessary to first establish the safety and immunogenicity of a prototype vaccine in a preliminary formulation. However, changes in responses to vaccine can be observed when scale-up manufacturing is done or practical formulations are produced (1,2). Finally, issues related to commercial development must be considered. In a free market, public health need and scientific rationale supporting the likelihood of success of a candidate vaccine will increase the chances that a new vaccine will attract the financial resources needed for its development to licensure and use as a public health tool.

GENERAL DESCRIPTION: PHASES OF CLINICAL TRIALS The clinical investigation of a new candidate vaccine progresses in three phases on the road to licensure. Although these phases are usually conducted sequentially, they may overlap. A phase I trial is the first human use of the vaccine candidate in healthy volunteers. Participants in phase I studies are typically closely monitored. These studies are designed to determine the frequently occurring, short-term side effects and the dose response to a candidate vaccine. In a phase I vaccine study, the immune response to the vaccine is measured; the analogous information in a similar study of a new drug would be its pharmacological characteristics in a small number of subjects. The information generated in phase I about the vaccine’s safety profile and immunogenicity should be sufficient to design expanded studies of safety and immunogenicity in phase II. Phase II studies are controlled, closely monitored studies of safety and immune response in an expanded number of subjects, perhaps several hundred. Some individuals who participate in phase II studies may represent the target population for which the test vaccine is intended. For example, infants or elderly subjects may be enrolled if the vaccine candidate is intended for ultimate use in these populations. Multiple phase II studies are often conducted to develop a database to direct the design of phase III studies.

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The development of a vaccine candidate can be accelerated if there is a human challenge model for the disease against which the vaccine is directed. This allows a preliminary assessment of vaccine efficacy (so-called phase IIb) by comparing disease attack rates in vaccinees and unvaccinated control volunteers. These challenge studies can be ethically justified if they are conducted by qualified investigators with rigorous adherence to a scientifically valid protocol and clear safeguards for the volunteers (3,4). It should be recognized that such challenge studies represent experimental models and may not exactly reproduce the disease as it occurs in an endemic area. For example, the inoculum used for challenge for diarrheal illnesses such as cholera and enterotoxigenic E. coli are probably higher than commonly occur in nature. This is to ensure that the attack rate among challenged volunteers is high enough to achieve statistical differences when comparing small numbers of vaccinated and unvaccinated individuals. However, the experimental challenge model is designed so that the challenge is not so rigorous as to overcome immunity; often the model has been tested by establishing that immunity induced by primary challenge-induced infection is not overwhelmed by a second challenge with the same pathogen (5). This level of immunity, that is, immunity after primary infection, is often the gold standard for immunity induced by vaccination. At the Center for Vaccine Development, University of Maryland, challenge models have been applied to testing vaccines against cholera, diarrheagenic E. coli, Shigella, Rocky Mountain spotted fever, malaria, influenza, and typhoid fever. If acceptable safety and immunogenicity are observed during phase II, phase III studies are planned to evaluate efficacy. Occasionally, phase III studies are designed to measure immunogenicity only. For example, if the protective immune response is known, then demonstration that most subjects attain that response after vaccination may be sufficient for licensure. Generally, a phase III study is a double-blind, controlled study of the new vaccine in a more heterogeneous population, under conditions more closely resembling those under which the vaccine may eventually be used. The study may include as a control group a true placebo, a licensed vaccine against another disease, or another licensed or experimental vaccine against the same disease. In a phase III study, the rate of occurrence of side effects that occur infrequently may be measured more accurately. Defined endpoints must be chosen, and a hypothesis stated. A sample size should be chosen on the basis of assumptions of the expected incidence of disease, and the reduction in disease incidence that is anticipated in vaccinees. Recently, phase III trials of rotavirus vaccines were specifically designed with large sample sizes to exclude the rare occurrence of an adverse event, intussusception. A pivotal study is a phase III study, which provides the most convincing data supporting the licensure of the vaccine. The pivotal protocol must be rigorously designed and analyzed with impeccable statistical considerations. After phase III studies demonstrating the safety and efficacy of the vaccine candidate, the sponsor of the vaccine who will market the product in the United States submits a Biologic Licensing Application (BLA) to the Food and Drug Administration (FDA). Approval requires that the safety and efficacy be demonstrated in well-designed, controlled studies. Once the application is approved, the vaccine may be sold commercially for the specific indication. After the BLA is approved, FDA requires that the holder of the BLA conduct postmarketing surveillance and submit periodic reports including incidence of

adverse reactions and follow-up of ongoing phase III studies. These data are generally descriptive in nature. After marketing approval, additional formal studies may also be designed to continue to measure efficacy and side effects. These studies, termed phase IV studies, may detect previously unknown, rare adverse reactions among recipients of the marketed vaccine. Many countries have formal systems in place to detect these events and determine using various epidemiologic methods whether there appears to be a relationship to vaccine administration. In the United States, for example, the Vaccine Adverse Event Reporting System (VAERS) and Vaccine Safety Data Link (VSD) systems are used for this purpose.

FACILITIES In general, facilities for vaccine testing include clinical office space for interviews for screening, obtaining consent, and conducting follow-up procedures; facilities for specimen collection and storage; and emergency equipment for treating anaphylactic reactions. The majority of studies can be conducted in an outpatient facility. In phase I and II studies of vaccines, unlike drug studies, participants usually take only one to three doses of the experimental agent. Many early studies of new vaccines require that signs and symptoms be recorded for a relatively short period after vaccination and that a limited number of blood tests be obtained to measure immune response over a period of weeks to months. The intensity of surveillance depends on the type of vaccine and the anticipated nature and incidence of side effects. Phase I studies of live vaccines in adults are usually conducted in an inpatient facility to collect preliminary safety data, and to determine the excretion of vaccine and potential for person-toperson transmission. For example, the degree of attenuation of some live enteric vaccines is unknown; these must be given under close inpatient supervision (6,7). Live vaccine studies may require frequent collection of stool samples or respiratory secretions for culture. In addition, for studies requiring very intense surveillance or frequent collection of specimens, inpatient studies may be required to ensure that every event (e.g., fever) is detected and recorded and to ensure compliance with collection of every specimen. For example, live oral Salmonella enterica serovar Typhi vaccine strain CVD 906 was found to be insufficiently attenuated, and to cause symptoms of typhoid fever in some inpatient volunteers (8). Concerns about the release of genetically engineered organisms into the environment before their preliminary safety and potential for personto-person transmission have been established requires that some studies be conducted on a closed isolation ward with strict contact isolation measures. In the United States, the National Institute of Allergy and Infectious Diseases supports several vaccine evaluation and treatment centers at academic institutions. Some of these centers have access to inpatient units where volunteers can be housed for intensive surveillance and specimen collection. In studies of experimental vaccines in children and infants, surveillance for adverse effects and collection of specimens is carried out in an outpatient setting. Telephone interviews with parents or guardians, collection of questionnaires filled out by parents, and review of medical records are means utilized to collect safety information. Children return to the clinic or physician’s practice for blood drawing or collection of respiratory secretions. Collection of stools can be accomplished either by instructing the caretaker to bring in soiled diapers or by sending a

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messenger to the home. Day care settings can be arranged for studies in which it is necessary to collect extensive clinical data or multiple specimens. The children are observed during the day by nursing staff, and they return home in the evening where the parents continue surveillance. This arrangement is sometimes optimal for phase I studies of live, attenuated vaccines.

REGULATORY ISSUES History of the Regulation of Vaccine Development The ancient Egyptians and Hebrews had strict meat handling laws, and later, ancient Greeks and Romans had regulations prohibiting the addition of water to wine. In the Middle Ages, grocers and druggists had trade guilds, which prohibited adulteration of drugs and spices. In the United States, there have been laws governing the size of a loaf of bread and prohibiting adulteration of bread; in 1785, the first comprehensive food adulteration law was enacted in the United States. In 1938, the Federal Food, Drug, and Cosmetic (FD&C) Act was enacted in the United States in response to a number of deaths caused by the use of diethylene glycol (anti-freeze) as the vehicle for an elixir of sulfanilamide. This act required sponsors of investigational new drugs (IND) to submit safety data about the candidate product before premarket approval. The turning point for modern regulatory affairs was the passage in 1962 of the Kefauver-Harris amendments to the FD&C Act. The 1962 amendments required that efficacy data, as well as safety data, be submitted to support IND applications. These amendments followed shortly after the discovery that thalidomide caused birth defects. Although thalidomide was never approved in the United States, it was being used extensively in research. Before the 1962 amendments, there was no requirement that FDA be notified of the use of investigational drugs or regulate their use. Today, sponsors of new vaccines must submit both safety and efficacy data to support the application. Since these data are gathered through clinical investigations, all sponsors must secure an IND and follow a set of principles known as Good Clinical Practice (GCP). GCP is the set of federal regulations and guidelines for clinical trials that will support an eventual application for licensure of a new vaccine or drug. GCP is designed to ensure the quality and integrity of clinical data and to protect the rights and safety of volunteers. GCP guidelines are described in detail in numerous Internet sites from various agencies, including the U.S. Department of Health and Human Services Office for Human Research Protection (OHRP), U.S. FDA, U.S. Army, U.S. Centers for Disease Control and Prevention, the International Conference on Harmonization (IHC), and others. These regulations are comprehensive, including protocol design and development, informed consent guidelines, record keeping, data reporting, adverse event reporting, etc. In the United States, when Congress passes a law, the regulatory agency involved writes the regulation and is responsible for enforcing the law. The Code of Federal Regulations (CFR) contains these regulations. Title 21 of the CFR deals with food and drugs, and Title 45 part 46 deals with protection of human subjects. These regulations give specific directions for all individuals—sponsors, monitors, and investigators— involved in a vaccine trial. The following parts of Title 21 are relevant to clinical investigations of vaccines: Part 50 (informed consent) Part 56 (institutional review boards)

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Part 312 (investigational new drug applications) Part 601 (licensing) Part 814 (pre-market approvals)

Elements of an Investigational New Drug Application The components of an IND application are described in 21 CFR part 312. An IND is filed for a vaccine that has never been approved in the United States; for a new dose, route, or schedule of administration of an approved vaccine, or for a new indication of an approved vaccine. The application includes a completed and signed form FDA 1571, which is a master administrative document with a table of contents that serves as a checklist for the elements of the application. The signature of the sponsor indicates that he/she agrees to conduct the investigation in accordance with all applicable regulatory requirements, specifically, to wait for 30 days after the FDA receives the IND before beginning the study, not to conduct the study if the study is placed on ‘‘clinical hold,’’ and agree to the review and approval of the study by an institutional review board (IRB). After the form 1571, there is an introductory statement about the vaccine’s characteristics, a general investigational plan, an investigator’s brochure, and the clinical protocol. Form FDA 1572 and the curricula vitae of the investigators are included. The form 1572 is a contract between the clinical investigator and the federal government to assure his/her compliance with 21 CFR 312, involving adherence to protocol, use of informed consent, record keeping, reporting, etc. Next are sections on chemistry, manufacturing and control information, pharmacology and toxicology information, and previous human experience. As the development of the vaccine progresses, the IND application is supplemented with protocol amendments, new protocols, new investigators, safety reports, information about microbiology or toxicology, and annual reports.

Obligations of Sponsors The sponsor of a clinical investigation is the person who has assumed responsibility for compliance with the FD&C Act and FDA regulations and guidelines. The sponsor submits and maintains the IND application. Not until the IND has been prepared can the investigational product be shipped for the purpose of conducting clinical trials. A sponsor who both initiates and conducts a clinical investigation is called a ‘‘sponsor-investigator.’’ The specific legal responsibilities of the sponsor, contained in 21 CFR, include selecting investigators, providing adequate information to investigators, monitoring investigations, ensuring compliance with proper IND procedures, and informing FDA and the investigators of any adverse effects or risks of the product being studied. Sponsors may transfer all or part of their obligations to a contract research organization (CRO). In 2004, the members of the International Committee of Medical Journal Editors issued a statement indicating that registration of clinical trials would be a requirement for subsequent publication of trial results (9). The purpose of registration is to provide results to study participants and to make public a list of all clinical trials. The Web site Clinicaltrials.gov is the information repository for posting information about clinical

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trials. Trials that must be registered include any project in which an intervention and a comparison group are studied to determine the effect of the intervention on a health outcome (9). Phase I trials have generally been excluded from the registry (10). It is the responsibility of lead sponsors, working together with investigators, to register clinical trials (11).

Obligations of Monitors The monitor of a clinical investigation confirms that the study is conducted according to the protocol developed by the investigator and sponsor, and according to FDA regulations. This is accomplished by meeting with the investigator and the research staff before a study begins and confirming the adequacy of the investigator’s facilities. The monitor makes periodic reviews of the investigator’s source documents, case report forms, and required reports. Problems with the study must be documented, and corrective actions taken.

Obligations of Investigators Similarly, an investigator’s responsibilities are contained in the FDA regulations. His/her agreement to conduct an investigation in accordance with regulations and the clinical protocol is documented when the investigator signs form FDA 1572, which is filed with the IND application. In 2000, the U.S. NIH issued a directive requiring that federally funded clinical researchers provide evidence of training on the protection of human research participants and on GCP. Briefly, the investigator must obtain IRB approval for the protocol, the consent document, and recruiting materials used to identify volunteers. He/ she must obtain approval for study amendments and file regular reports with the IRB. The investigator must keep immaculate records and must report serious and unexpected adverse events to the sponsor and the IRB. The investigator must administer the vaccine and maintain records accounting for the product disposition. He/she is responsible for educating volunteers and obtaining written informed consent before volunteers become involved in the study. The investigator is obligated to store records, and allow FDA representatives to inspect the study records.

Institutional Review Boards An IRB is a group designated by an institution to review and approve biomedical research involving humans. IRBs are responsible for the well being of subjects involved in clinical trials. The board includes at least five members, at least one who is not a scientist, and one who is not affiliated with the institution. The IRB reviews protocols, investigator’s brochures, consent forms, recruiting materials, and additional safety information. The membership of an IRB, standard operating procedures, review of research, voting and quorums are defined in part 56 of 21 CFR.

Record Keeping and Product Accountability for Clinical Trials Compulsive record keeping is an important component of GCP. The need for privacy and protection of medical records has led to regulations under the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Restraints on the use of medical information for research were imposed in 2003. The new privacy rules are under debate as regulators attempt to

balance the public interest in research with the public interest in privacy (12,13). Both investigators and sponsors should retain the same records. Case report forms are uniform at all the sites conducting the study, and allow the sponsor to look at the same information in the same format from different sites. The case report forms may be used for data entry and analysis and should be designed to efficiently capture the data points that precisely correspond to the aims and endpoints of the protocol. ‘‘Source documents,’’ those records on which the information about a participant is first recorded, may be used for some studies. The type of information to be collected may vary with the protocol, but in general it would include subject identification, protocol name and number, sponsor’s name, date of participant’s visit, procedures and tests completed, concomitant medications, occurrence of adverse experiences, and the name of the person entering the information and the date. Corrections to the study records must be initialed and dated. In 1997, the U.S. FDA established regulations about the use of electronic records in clinical trials (21 CFR part 11). The regulations permit use of electronic signatures. Investigators have the option of maintaining records as paper files or electronic files. The electronic record must provide an audit trail, that is, a record of who enters or changes data and when (14). Teleforms for electronic database entry and on-line case report forms have replaced paper forms for most studies. Investigators usually develop a protocol-specific quality management plan to ensure the correctness of the data collected. A sponsor should have a policy about monitoring case report forms that indicates how frequently forms are to be monitored and how intensively. Monitors compare source documents with case report forms, looking for inconsistencies, errors, and appropriate signatures. In addition to maintaining clinical records, investigators are required to maintain records for the receipt and disposition of the experimental product. The records should include the name of the material, its IND number, its condition, the lot number, date, and source. Records should show the name of persons who received the study vaccine and what was done with extra doses. Each dose must be accounted for. To assure that the experimental product is not tampered with, vaccine materials should be stored in a secure refrigerator, freezer, or cabinet.

Reporting Adverse Experiences The NIH has developed policies for safety monitoring of all studies that evaluate investigational drugs and biologics. These policies require that there is a system for oversight and monitoring of clinical trials. The mechanism of oversight depends on the risk and complexity of the trial and may be a full data and safety monitoring board, a safety committee, or an individual independent safety monitor. FDA regulations require investigators to report all adverse experiences to the sponsor of a study. If the experience is serious or unexpected, the event must be reported promptly to both the sponsor and the responsible IRB. A serious adverse experience is ‘‘any experience that is fatal or life-threatening, is permanently disabling, requires or extends inpatient hospitalization, or is a congenital anomaly, cancer, or overdose.’’ An unexpected adverse experience is ‘‘any adverse experience that is not identified in nature, severity, or frequency in the current investigator brochure; or, if an investigator brochure is

Chapter 3: Initial Clinical Evaluation of New Vaccine Candidates

not required, that is not identified in nature, severity, or frequency in the risk information described in the general investigational plan or elsewhere in the current application’’ (21 CFR 312.32). As a general rule, many sponsors require investigators to report all adverse experiences even if the event is not apparently related to the vaccine. The investigator must keep a record to indicate the treatment and outcome of the adverse experience.

Regulatory Considerations in International Trials International trials are of particular significance in vaccine development, in which the ultimate target population may be individuals in a country other than that in which the vaccine was manufactured. Such trials may reveal differences in safety, immunogenicity, and efficacy when the vaccine is studied in a new population. For example, a live oral cholera vaccine was less immunogenic when given to Thai adults than to U.S. adults (1), and oral polio vaccine was less immunogenic in children in developing countries (15). The experimental vaccine may or may not be studied under U.S. IND. If not, the vaccine must be manufactured outside the United States. Several provisions must be met for the U.S. FDA to accept data from an international trial. These include (i) the data must be applicable to U.S. populations; (ii) international investigators must be competent; (iii) the protocol must be reviewed for ethical considerations; and (iv) the site must be available for FDA inspection. The same standards that apply to studies in the United States should be used in studies in foreign countries (16). The research should be developed in close collaboration with local investigators and other authorities in the country in which it will be performed (17). FDA does not require that case report forms or source documents be completed in English, but a translator may be required if the site or records are inspected. Local customs may affect several aspects of the trial, such as the means of obtaining and documenting informed consent and the recognition and reporting of the types of experiences that are considered adverse.

PROTOCOL DEVELOPMENT General Considerations The success of a vaccine trial in phase I or II is largely predicted by the quality of the protocol. According to the CFR, a protocol must contain the following components: (i) a statement of the purpose and objectives of the study; (ii) the name and address of the investigator, the name and address of the research facilities, and the name and address of the reviewing IRB; (iii) a statement of the number of participants and the inclusion and exclusion criteria for participating; (iv) the study design, including the type of control group, if any; (v) the dose to be given and method for determining the dose; (vi) a description of the outcomes to be measured; and (vii) a description of the measures to be taken to monitor the participants and to reduce risks. In addition, many protocols contain a discussion of the scientific background and rationale to place the study in context. It is important to include information about the disease, its clinical nature and epidemiologic importance, and whatever is known about the elements of protective immunity. This is useful in justifying the need for the study and risk to volunteers. The type of study, for example, controlled, double- or single-blinded, and the method of randomization, if any,

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should be included. Outcomes to be observed need to be clearly described; objective definitions of outcomes are highly desirable. Definitions of safety (e.g., degree of temperature that defines fever) and immunogenicity (e.g., definition of seroconversion) need to be clearly decided and documented during protocol development. A justification for the dosage should be provided. The means of monitoring patients and contingencies for handling side effects should be described. Protocols include a section describing the statistical tests to be used to analyze the results and a section to justify the sample size chosen. In phase I studies, however, it is usually not possible to detect statistically significant differences between groups because of the small numbers of participants.

Considerations for Studies Involving Children and Infants In designing a protocol to be carried out in children, additional considerations are required. In 1998, NIH issued a policy and guidelines on the inclusion of children as participants in research involving human subjects, providing guidance on inclusion of children and justification for exclusion of children in research funded by NIH. In considering the inclusion of children in vaccine studies, the first decision to be addressed is the age group to be vaccinated. The answer depends on the age at which children are at risk for the infection the vaccine is designed to prevent. For most pathogens, it is optimal to provide protection as early in life as possible. However, the presence of small amounts of maternal neutralizing antibody may inactivate some live viral vaccines, such as measles vaccine, requiring that immunization be postponed to a later age. Usually, pediatric vaccine development proceeds in older children, and progresses step-wise to younger children until the target age group is reached. Early infancy is a time when children receive multiple routine vaccinations. An important issue is whether or not to give an experimental vaccine at the same visit with licensed vaccines. Frequently, phase I studies will dictate a four-week separation between the study vaccine and any other vaccinations, to avoid either confounding the safety data or inducing immune interference with simultaneously administered vaccines. To be logistically practical and economically feasible, new vaccines should eventually be incorporated into the routine vaccination schedule of infancy. Therefore, the effects on safety and immunogenicity of concurrent immunization should be evaluated in phase II studies. The number of doses of vaccine to be administered must also be determined. Two or more doses are often necessary to overcome maternal antibody or induce priming. This issue is most commonly addressed by giving two or three doses and measuring antibody levels before and after each immunization. The necessity, practicality, and ethics of including a placebo group should also be carefully weighed. In designing a protocol for pediatric studies, one must carefully balance the need to be minimally invasive but to collect all necessary data. This sometimes requires compromises. The most difficult aspect of carrying out a successful pediatric vaccine trial is recruitment of sufficient numbers of children. Parents are protective of their children and will refuse to enroll their children or continue to participate in a study that they perceive is too invasive, or involves undue discomfort for their child. The number of times that specimens are sampled, therefore, should be kept to a minimum.

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Considerations for Vaccines Prepared by Recombinant DNA Technology or Containing Recombinant DNA The development of attenuated viral and bacterial strains for use as vaccines was one of the obvious applications of the techniques for recombining DNA discovered in the mid-1970s. Before this time, live viral (18) and bacterial (19) vaccine strains were developed by repeated in vitro passage or by chemical mutagenesis, techniques that resulted in undefined mutations. Nevertheless, live vaccines against diseases such as measles, mumps, rubella, and typhoid fever were developed and licensed. Recombinant DNA technology offered the means to develop attenuated vaccines in which the precise molecular mechanism of attenuation could be known. Despite the precision of molecular DNA techniques, vaccines developed using recombinant DNA were thought by some to be threatening to the natural environment. To document the potential for environmental consequences of vaccinating humans with such vaccines, sponsors are required to include an environmental analysis (21 CFR 312.23), which includes justification for a claim for categorical exclusion or an environmental assessment. Such justification might include data showing the survivability of the vaccine strain in various natural environments such as local water, soil, and food, especially in comparison with the wild-type pathogen (20). Phase I protocols to study the safety of recombinant vaccines generally contain provisions for studying the potential for person-to-person transmission of these strains.

Considerations for Vaccines that Can Be Transmitted Person-to-Person Many of the currently used live bacterial and viral vaccines are shed in respiratory secretions (e.g., live attenuated influenza vaccine) or stool (e.g., polio vaccine), and are potentially transmitted person-to-person (21). Transmission of oral polio vaccine was considered desirable in the early years of its use, since such transmission led to herd immunity (22). Transmission to pregnant women or immunocompromised individuals is now recognized as a risk of the use of live vaccines that can be spread from person-to-person, for example, transmission of vaccinia virus or its recombinant virus to an individual with eczema can result in severe vaccinia infection (23). As a result of this concern, many phase I and II clinical protocols include preliminary measurements of the potential for person-to-person spread of live vaccine candidates. Initially, this may require that the vaccine strain be studied in isolation until a gross assessment of its transmissibility is established, for example, among unvaccinated adults residing with vaccinees on a research isolation ward. Examples of such studies to assess person-to-person transmission include studies of S. typhi vaccine strain CVD 908-htrA (24), Shigella vaccine strain 1208S (25), and of a recombinant vaccinia virus expressing gp160 of HIV (26). In phase II, volunteers who reside with infants, pregnant women, or immunocompromised individuals may be excluded because of the possibility of transmission of a vaccine strain whose safety is not completely established. phase II studies of transmissibility might include cultures of the stool or respiratory secretions of household contacts of vaccinees, and in later phases, attempts at vaccine isolation from environmental reservoirs such as sewage. The testing of live oral cholera vaccine strain CVD 103HgR is a good example of how such testing is executed. This

V. cholerae O1 strain is deleted in 94% of the toxic A subunit of cholera toxin (27). In phase II clinical studies, the possibility of transmission of this strain to contacts of vaccinees and to the environment around the households of vaccinees was investigated (28,29). In brief, this strain was shed for a short period by only a small proportion of vaccinees, was minimally transmitted to contacts of vaccinees, and was not recovered from the natural environment near vaccinees.

SELECTION OF VOLUNTEERS General Considerations Initial phase I studies of candidate vaccines generally involve healthy adult volunteers, that is, those who have no abnormality that would confound the interpretation of the safety of the product or increase the likelihood of their having an adverse event. Healthy volunteers may be recruited from the community at large, or interested students, or employees at research institutions. Students and employees can be a vulnerable population, however, and care must be taken to ensure that there is no element of pressure or coercion to participate. In addition, some protocols may have a seroeligibility requirement, usually the absence of serum antibody to a particular antigen. Rarely, a protocol may specify that only individuals of a certain human leukocyte antigen (HLA) type may participate, when preclinical data indicate that immune responses will be restricted to a certain genotype. The protocol generally indicates what tests must be performed to establish volunteers’ health. For example, some or all of the following may be done: medical history, physical examination, complete blood count, serum chemistries, urinalysis, HIV serology, and pregnancy test. Previously, women of childbearing potential were sometimes precluded from participation. However, in 1993, guidelines for the study and evaluation of gender differences in the clinical evaluation of drugs were issued, stating that women be included provided appropriate precautions against becoming pregnant are taken, and that women are counseled about the importance of these precautions. Efforts should be made to ensure that women participants are not pregnant at the time of enrollment, and that women are informed about animal reproduction studies and teratogenic potential of the vaccine. Generally, however, such data are not available for experimental vaccines. In 2001, the U.S. Department of Health and Human Services released additional protections pertaining to research in pregnant women. In these additional regulations, there must be direct benefit to the woman or fetus as a result of the research, or there must be only minimal risk to the fetus, and the new information learned in the research cannot be obtained in any other way. Phase II vaccine studies also involve healthy adults. Once preliminary safety has been established in the phase I study, the screening to demonstrate the health of volunteers may be less rigorous. For example, the following may be done: medical history, complete blood count, HIV serology and pregnancy test. The recent sequencing of the human genome has provided the opportunity to better understand the variations in safety and immune responses observed among apparently similar healthy individuals enrolled in vaccine trials. The importance of genotype on predicting response to drugs (pharmacogenomics) is well established, and these principles are beginning to be applied to understanding the variable occurrence of adverse events and immune responses to vaccines, so-called

Chapter 3: Initial Clinical Evaluation of New Vaccine Candidates

vaccinomics (30). The variability in immunity results largely from differences in genes that control the immune responses. Variable immune responses depend on HLA genes, cytokine and cytokine receptor genes, killer cell immunoglobulin-like receptors, genes of the leukocyte receptor cluster, signaling molecules, the natural resistance-associated macrophage protein-1 gene, and many others (30). As new vaccines are developed and tested in the future, understanding the influence of polymorphisms in immune response genes will be critical to designing safe and effective vaccines and predicting vaccine efficacy. One might predict that in the future, vaccines will be designed that are expected to be safe and immunogenic in people of defined genotypes, and that vaccine testing will involve enrolling volunteers based on their genomic makeup.

Considerations for Studies Involving Children and Infants Pediatric vaccine trials are carried out in healthy children who have no personal or family history of immune deficiency. The exception to this would be a vaccine targeted for a particular population, such as a Pseudomonas vaccine for children with cystic fibrosis. Screening of healthy children generally involves only a medical history and physical examination. Specific baseline data may be collected if there is specific concern about potential vaccine side effects; for example, liver function tests are performed if a live viral vaccine might cause hepatitis. Transmission of live viral vaccine strains to contacts through the stools or respiratory secretions is a particular concern in studies involving young children. Until it is demonstrated that the vaccine virus is not transmitted, initial live viral vaccine studies should not include children attending group day care or children residing in a household with an immunosuppressed individual. Recruitment of children into vaccine trials is usually carried out through outpatient settings providing well child care, such as private practices, hospital clinics, and health maintenance organizations. The optimal method of approaching families varies, depending upon the population served and the setting. In many centers, a study nurse and/or investigator approach the parent or guardian on the day they wish to enroll the child. Sending literature to the parents before the vaccine visit provides an important opportunity for the family to discuss the study, and to contact the study personnel by telephone for further information before they must make the decision whether or not to participate.

CONSENT General Considerations The FDA regulations concerning informed consent are contained in 21 CFR Part 50. Consent is not an endpoint but a continuing communication between participant and investigator during which the participant receives all the information he/she needs to participate in the study. The process should include ample opportunities for the free exchange of information and for the participant to ask questions. Consent should be obtained under circumstances that give the potential participant the opportunity to carefully consider the decision to participate with no coercion or undue influence. Such features as the place, the time, and the person who provides the information may affect a subject’s ability to make an informed judgment. Some investigators use a formal mechanism to assess

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whether the volunteer adequately understands the key elements of the study. This could be a question and answer session, or a written test in which the volunteer must answer a specified proportion of questions correctly in to qualify for participation. The principles of informed consent include the following: (i) the purposes, procedures, and experimental nature of the protocol are described fairly; (ii) the discomforts and risks to be expected are described; (iii) information about appropriate alternative procedures (for vaccine studies, this might be information about the existence of a licensed vaccine for the same disease); (iv) information about whom to ask for further information; and (v) the statement that an individual is free to withdraw his/her consent and discontinue participation without prejudice. It is key that the information provided be understandable to the participants. This means that the information be presented in the participant’s language and that technical and medical terms be explained or replaced with lay terms appropriate to the participant’s level of education. Often a consent form can be simplified by using short, declarative sentences. In addition, the consent document should not include statements that release the investigator from responsibility or that waive the volunteer’s rights. Consent must be documented in writing. The FDA requires that the IRB reviews and approves advertisements and other materials used to recruit participants. Recruiting materials are considered an extension of the consent process. These materials, such as advertisements and fliers, should not be misleading. FDA recommends that the advertisement include only the following: (i) name and address of the investigator; (ii) purpose of the research and a summary of eligibility criteria; (iii) a description of the benefits (including payment); and (iv) the location of the research and person to contact for more information. It is important to avoid making claims about the vaccine. It is also important that the payment of volunteers not be so much as to affect the ability of the volunteer to assess risks and benefits appropriately. Few research groups or organizations have specific standards (31–33). Volunteer compensation scales should be carefully conceived to ensure that economically disadvantaged individuals are not unduly influenced by the financial compensation offered. This concern applies not only to economically disadvantaged individuals, but students and middle class populations as well. Dickert and Grady describe three approaches related to volunteer payments (34). The first approach is the market model, which is grounded in the freemarket principle that supply (availability of interested and eligible volunteers) and demand (the investigator’s desire to complete a trial with a specified number of subjects within a defined time frame) determine how much subjects should be paid for participation. A second model is the reimbursement model, in which payment is provided simply to cover volunteers’ expenses (travel, meals, parking, child care), similar to jury duty payments, such that the volunteers accrue no profit, thereby minimizing financial inducement. The third and most accepted model is the wage payment model. This model purports that participating in research is similar to many other forms of unskilled labor in that it requires little skill and training, but may involve some risk. In this model, subjects are paid for work that is valuable to society, on the basis of a standard wage for unskilled labor. In general, volunteers should have characteristics that make them suitable for other jobs in the community, particularly entry-level jobs, to ensure that the decision to participate in research is truly optional.

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Investigators who have conflicts of interest should make these known to volunteers in the consent form. A conflict of interest exists when an investigator has financial or personal relationships that inappropriately influence his/her actions or judgment (35).

Considerations for Studies Involving Children and Infants Children are not empowered to grant informed consent until they have reached the legal age of majority, or have been deemed emancipated. The degree of involvement of a child in the decision to participate in a vaccine trial depends on his/her intellectual capacity and stage of development. Assent to participate usually should be obtained from children with an intellectual age of seven years. In trials involving children, a parent or guardian assumes responsibility for the child, and grants consent by proxy. This places an increased responsibility on the sponsor, the investigator, and the IRB to assure that risk is minimized, the study is fully understood by the parent, and that there is no coercion to participate. It is essential that the parent understand that participation is entirely voluntary, and that the parent may withdraw the child from the study at any time. An investigator who also serves as the child’s primary care physician has the additional burden to assure that the parent does not feel compelled to enroll the child to please the physician. Remuneration for participation is a more sensitive issue in trials involving children than adults. Rewards must be only a token of appreciation, and not of a magnitude to induce parents to enroll the child into the study. Generally acceptable compensation includes a small savings bond, free routine vaccinations, or payment for costs incurred by the trial, such as travel.

6.

7.

8.

9.

10.

11. 12. 13. 14. 15.

16.

17. 18.

SUMMARY Vaccine testing enters the twenty-first century using methods based on sound epidemiological and ethical principles by which the safety and efficacy of future vaccines will be established. Although vaccine development may be an empirical science, the methodological framework for clinical testing to determine safety and efficacy has been largely codified. Precedents have been set for the study of vaccines containing recombinant DNA and for methods to study the transmissibility of live vaccines. We anticipate that clinical vaccine testing will continue to be a productive and exciting area of clinical research as vaccines against infectious diseases, such as malaria, tuberculosis, HIV, and agents of bioterror, are developed.

19.

20.

21.

22.

23.

REFERENCES 1. Su-Arehawaratana P, Singharaj P, Taylor DN, et al. Safety and immunogenicity of different immunization regimens of CVD 103HgR live oral cholera vaccine in soldiers and civilians in Thailand. J Infect Dis 1992; 165:1042–1048. 2. Levine MM, DuPont HL, Hornick RB, et al. Attenuated, streptomycin-dependent Salmonella typhi oral vaccine: potential deleterious effects of lyophilization. J Infect Dis 1976; 133:424–429. 3. Miller FG, Grady C. The ethical challenge of infection-inducing challenge experiments. Clin Infect Dis 2001; 33:1028–1033. 4. Rosenbaum JR, Sepkowitz KA. Infectious disease experimentation involving human volunteers. Clin Infect Dis 2002; 34:963–971. 5. Kotloff KL, Nataro JP, Losonsky GA, et al. A modified Shigella volunteer challenge model in which the inoculum is administered

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with bicarbonate buffer: clinical experience and implications for Shigella infectivity. Vaccine 1995; 13:1488–1494. Levine MM, Kaper JB, Herrington D, et al. Safety, immunogenicity and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103-HgR. Lancet 1988; 2:467–470. Hohmann EL, Oletta CA, Killeen KP, et al. phoP/phoQ-deleted Salmonella typhi (Ty800) is a safe and immunogenic single-dose typhoid fever vaccine in human volunteers. J Infect Dis 1996; 173:1408–1414. Hone DM, Tacket CO, Harris A, et al. Evaluation of a double aro mutant of Salmonella typhi ISP1820 in volunteers and in U937 human macrophage-like cells. J Clin Invest 1992; 90:412–420. DeAngelis C, Drazen JM, Frizelle FA, et al. Clinical trial registration: a statement from the International Committee of Medical Journal Editors. Ann Intern Med 2004; 141:477–478. DeAngelis C, Drazen JM, Frizelle FA, et al. Is this clinical trial fully registered? A statement from the International Committee of Medical Journal Editors. Available at: http://www.icmje.org/ clin_trial up.htm. PRS Information. Registration of clinical trials. Available at: http://prsinfo.clinicaltrials.gov/. Kulynych J, Korn D. The effect of the new federal medical-privacy rule on research. N Engl J Med 2002; 346:201–204. Annas GJ. Medical privacy and medical research—judging the new federal regulations. N Engl J Med 2002; 346:216–220. DeMarinis AJ, Tandy MK. 21 CFR Part 11: electronic records and signatures—the shock of it! Res Pract 2000; 1:157–182. Patriarca PA, Wright PF, John TJ. Factors affecting the immunogenicity of oral poliovirus vaccine in developing countries: review. Rev Infect Dis 1991; 13:926–939. Shapiro HT, Meslin EM. Ethical issues in the design and conduct of clinical trials in developing countries. N Engl J Med 2001; 345:139–142. Varmus H, Satcher D. Ethical complexities of conducting research in developing countries. N Engl J Med 1997; 337:1003–1005. Lavergne B, Frappier-Davignon L, Quevillon M, et al. Clinical trial of Trivirix for measles, mumps, and rubella immunization. Canad Dis Wkly Rep 1986; 12:85–88. Germanier R, Fure¨r E. Isolation and characterization of galE mutant Ty21a of Salmonella typhi: a candidate strain for a live oral typhoid vaccine. J Infect Dis 1975; 141:553–558. Cryz SJ Jr, Kaper J, Tacket C, et al. Vibrio cholerae CVD 103-HgR live oral attenuated vaccine: construction, safety, immunogenicity, excretion and non-target effects. In: Non-Target Effects of Live Vaccines. Vol 84. Dev Biol Stand. Basel, Switzerland: Karger, 1995:237–244. Levine MM. Non-target effects of live vaccines: myth, reality and demagoguery. In: Non-Target Effects of Live Vaccines. Vol 84. Dev Biol Stand. Basel, Switzerland: Karger, 1995:33–38. Sabin AB. Oral poliovirus vaccine: history of its development and prospects for eradication of poliomyelitis. JAMA 1965; 194:872–876. Lane JM, Ruben FL, Neff JM, et al. Complications of smallpox vaccination, 1968. N Engl J Med 1969; 281:1201–1208. Tacket CO, Sztein MB, Losonsky GA, et al. Safety and immune response in humans of live oral Salmonella typhi vaccine strains deleted in htrA and aroC aroD. Infect Immun 1997; 65:452–456. Kotloff KL, Simon JK, Pasetti MF, et al. Safety and immunogenicity of CVD 1208S, a live, oral DguaBA Dsen Dset Shigella flexneri 2a vaccine grown on animal-free media. Hum Vaccin 2007; 3:268–275. Cooney EL, Collier AC, Greenberg PD, et al. Safety of and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein. Lancet 1991; 337:567–572. Kaper JB, Lockman H, Baldini MM, et al. Recombinant nontoxinogenic Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature 1984; 308:655–658. Simanjuntak CH, O’Hanley P, Punjabi NH, et al. The safety, immunogenicity and transmissibility of single-dose live oral

Chapter 3: Initial Clinical Evaluation of New Vaccine Candidates cholera vaccine CVD 103-HgR in 24 to 59 month old Indonesian children. J Infect Dis 1993; 168:1169–1176. 29. Gotuzzo E, Butron B, Seas C, et al. Safety, immunogenicity, and excretion pattern of single-dose live oral cholera vaccine CVD 103HgR in Peruvian adults of high and low socioeconomic levels. Infect Immun 1993; 61:3994–3997. 30. Poland GA, Ovsyannikova IG, Jacobson RM, et al. Heterogeneity in vaccine immune responses: the role of immunogenetics and the emerging field of vaccinomics. Clin Pharm Ther 2007; 82:653–664.

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31. Dickert N, Ezekiel E, Grady C. Paying research subjects: an analysis of current policies. Ann Intern Med 2002; 136:368–373. 32. Newton L. Inducement, due and otherwise. IRB 1982; 4:4–6. 33. Macklin R. On paying money to research subjects. IRB 1981; 3:1–6. 34. Dickert N, Grady C. What’s the price of a research subject? Approaches to payment for research participation. N Engl J Med 1999; 341:198–203. 35. Davidoff F, DeAngelis CD, Drazen JM, et al. Sponsorship, authorship, and accountability. N Engl J Med 2001; 345:825–827.

4 Special Issues in Performing Vaccine Trials in Developing Countries Milagritos D. Tapia and Karen L. Kotloff Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Christopher V. Plowe Howard Hughes Medical Institute and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Samba O. Sow Centre pour le De´veloppement des Vaccins-Mali (CVD-Mali), Bamako, Mali

INTRODUCTION Despite bearing the highest burden of most vaccine-preventable diseases, developing countries have, until recently, rarely served as sites for clinical trials of new and investigational vaccines. The failure to conduct vaccine trials in developing countries in a timely fashion in the past has contributed to huge disparities in the availability of routine vaccines, and many lives lost. With increasing appreciation of the promise of vaccines against diseases that occur primarily in developing countries (e.g., malaria) (1), these populations stand to benefit more rapidly as more vaccine trials are conducted in the developing world (2). This trend arises in part from recognition that risk-benefit assessments can differ in industrialized and developing country populations (3). Immunogenicity of a vaccine in an industrialized country population may be greater than that observed in other populations (e.g., oral cholera vaccine) (4,5). Moreover, some vaccines, as in the case of a conjugate vaccine against meningococcal A disease (6,7), are developed specifically for use in developing countries, and the clinical trials are being conducted primarily in these populations. Another impetus to conduct these trials in developing countries has been the availability of funding aimed at accelerating the introduction of potentially high-impact vaccines (e.g., rotavirus vaccine) into developing country routine vaccination programs (8,9). Nowadays these trials are often supported by partnerships between public and private agencies interested in addressing a public health problem in the developing world (6,8). Finally, as developing country governments decide whether or not to introduce new vaccines, data regarding the burden of vaccine-preventable diseases and the safety and immunogenicity of a particular vaccine that were collected in their regions are very influential locally as well as to potential donors. Without convincing evidence of a significant disease burden and results of a clinical trial in their region that demonstrates vaccine effectiveness under local conditions, governments of developing countries may be reluctant to commit their limited financial and other resources to introduction of a new vaccine, despite recommendations of

international technical agencies. Thus, it is important to perform clinical trials of vaccines in developing countries. This chapter will address the issues facing investigators and sponsors in the conduct of vaccine trials in the developing world. While the ethical issues surrounding the conduct of clinical trials in developing countries are addressed elsewhere, this chapter will address the logistical implications.

ISSUES RELATED TO TRIAL SITE PREPARATION While there are strong justifications for conducting vaccine trials in developing countries, there are special issues to consider along the path to success. The first is the development of a vaccine trial site with the appropriate infrastructure and expertise to conduct clinical research; this is a gradual process that begins months to years prior to any study and builds on the site’s previous experience. In this regard, sponsors may make a substantial investment toward the training of local personnel and provision of the necessary equipment. In addition, the international research community cooperates through their participation in multicenter trials and trial networks.

INFRASTRUCTURE Within the context of a vaccine trial, ‘‘infrastructure’’ refers to the framework that is in place to support the required activities, including personnel with clinical and laboratory expertise and the necessary equipment or hardware and physical structures in which to conduct trial activities. When all of these components are not immediately available, investigators should incorporate capacity-building activities into the preparation for the trial.

Personnel All study staff should be trained as researchers as well as in the procedures related to the trial. Training in international standards of the ethical conduct of research, as well as in the regulatory aspects of vaccine trials included in policies such as

Chapter 4: Special Issues in Performing Vaccine Trials in Developing Countries

the International Conference on Harmonization Guidelines, is essential for personnel. If personnel have not had this training, then the investigator is responsible for securing support for it from the sponsor or other supporting agency when planning the research. These activities contribute to sustainable research capacity. Given the variable levels of expertise among personnel, training can be customized to the level of study involvement. For example, training for personnel whose sole responsibility is to conduct household visits is likely to be limited to research ethics, focusing on confidentiality issues, whereas investigators and study coordinators who are responsible for supervising staff with a broad range of duties should be trained in research ethics as well as good clinical practices (GCP). Laboratory tests in relation to a vaccine trial have several purposes. Clinical safety laboratory testing is required in most phase I and II trials, in which hematological and chemical parameters may be necessary to detect toxicity. In this case, testing must be done locally as results are relevant to a volunteer’s enrollment and continued participation in the trial. Clinical laboratory assays that assess vaccine safety must be performed under the guidelines and tenets of GCP, since the results will be used as study end points and will be reviewed by national regulatory agencies. Training laboratory staff to this level of rigor can be challenging and requires regular quality control and assurance as well as external monitoring. GCPcompliant laboratory testing also challenges local investigators as they must obtain specialized equipment, secure a reliable supply of reagents that can be validated to perform the necessary assays and maintain rigorous record keeping. Certification by the College of American Pathologists or other recognized bodies for clinical assays is recommended, if possible. The term ‘‘good laboratory practices’’ (GLP) refers to a set of strict codified guidelines relevant for preclinical laboratory tests and animal studies. GLP guidelines do not apply to laboratory tests performed in relation to clinical trials. Nevertheless, GCP-compliant laboratory testing similarly involves rigorous and detailed standard operating procedures, use of unexpired reagents, and maintenance of meticulous records. Immunogenicity assays are also performed during a vaccine trial. Often sponsors prefer that these tests be performed in internationally recognized reference laboratories. However, where capacity exists, the local laboratory personnel may benefit from learning to perform this testing to the extent possible. For example, enzyme-linked immunosorbent assays can often be performed locally (sometimes with commercially available standardized kits), while highly specialized testing such as measurements of cell-mediated immunity may have to be performed elsewhere. Local performance of some laboratory assays builds capacity and ensures that at least a portion of the clinical samples are tested in country, which is often desirable to local ethics committees and investigators. Personnel are trained in a new assay and the local laboratory becomes better equipped with any materials the assay may require. Local laboratory scientists may travel abroad for training in specific techniques and to learn about the preventive maintenance of laboratory equipment.

Physical Infrastructure Cold chain maintenance for both vaccines and clinical samples, including vaccine transport mechanisms, is an important element of the site infrastructure. An intact cold chain is essential for maintaining the integrity of the study vaccine. A parallel

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cold chain is necessary to preserve clinical samples that will be tested for immunogenicity using sera and cells, and to characterize pathogens or vaccine strains (e.g., cryopreserved malaria parasites, nasal and throat washes for respiratory pathogens and vaccines, stool for diarrheal pathogens and enteric vaccines, etc.). In developing countries, especially in rural settings, maintaining a cold chain for vaccine storage and transport to the study site can be challenging. In many cases, the national Expanded Program on Immunization (EPI) officers can offer advice on how to anticipate and overcome local obstacles. Challenges may be weather related, such as monsoons in Asia or the African rainy seasons, which can affect travel and transport in country by impeding passage to certain areas. The extreme heat encountered in some areas necessitates a particularly robust and reliable cold chain that must be initially field tested under local conditions. The cold chain must be carefully maintained and backup equipment (refrigerators, freezers, redundant temperature monitoring systems) should be locally available to replace failed equipment without delay while repairs are made. Service contracts, while expensive, are necessary to maintain electrical equipment such as deep freezers and air-conditioning systems. Additional environmental concerns such as dust and humidity should be considered when devising equipment maintenance programs. Special measures such as custom-made covers to protect equipment can be locally designed and fabricated. Even where there is an existing EPI cold chain system, these are typically insufficient for the purposes of maintaining and documenting cold chains for investigational vaccines and important clinical samples. For example, vaccine storage as well as operation of clinical laboratory equipment typically requires air-conditioning, which entails a large power demand; preservation of peripheral blood mononuclear cells for measures of cell-mediated immunity requires 808C freezers and liquid nitrogen storage and transport capability; and processing samples may require expensive centrifuges, incubators, and other sensitive and expensive equipment that can be very challenging to maintain in good operating condition in harsh, remote environments. The possibility of loss of vaccine (or adjuvant) due to temperature deviations should be anticipated in planning the timing and size of vaccine production lots. When timing of the trial is critical, for example, malaria or meningococcal vaccine clinical trials that must be timed to a rainy or dry season, vaccines should be shipped with enough lead time so that new shipments can be sent to replace lost product. Electrical and water supply in developing countries can be tenuous; challenges include both power surges and failures, water outages during the dry season or flooding during rainy season, or simply a complete lack of power or water. Power surges and failures can adversely affect vaccine storage at any time of day, and maintenance programs should include the provision of surge protectors, uninterruptible power supply devices and generators, as well as reliable 24-hour vigilance to ensure backup power during failures. In rural settings, solar panels may be used as a power supply, although the power requirements of air-conditioners and freezers limit the usefulness of solar power sources. At a malaria vaccine testing site in Bandiagara, Mali, where municipal power is subject to intermittent outages, two paired sets of large diesel generators were installed to support vaccine trials. A malaria vaccine trial site in rural Burkina Faso runs solely on diesel-generated electricity and pumps its own water into a tower reservoir because there

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is neither municipal power nor water. These infrastructure elements are expensive, and may create problems by appearing ostentatious in settings of extreme poverty. Sponsors should exercise judgment and consider equity in making decisions about upgrading infrastructure. For example, if improvements are made to assure proper vaccine storage and the integrity of data entry, it is also advisable to invest in improving clinical examination rooms, waiting rooms, and health worker office areas to assure the comfort of subjects and investigators.

Environment Completing the follow-up of clinical trial participants in developing countries can also be challenged by the environment. In rural settings, transportation to and from study sites can be difficult for both study participants and personnel. In urban settings, populations tend to be more mobile, within cities or even between urban and rural areas, resulting in rates of loss to follow-up as high as 40% or more in some urban clinical trials with extended follow-up (10). This problem is especially pertinent in areas where there is seasonal migration. For example, during rainy seasons, persons may migrate to the agricultural fields seeking employment or to work on family farms. These migrations can often be anticipated and should be discussed at the time of recruitment so that study participants understand the timing of study procedures and any implications that may have on their travel plans. In rural settings, flooding during the rainy season may make access to homes of study participants impossible without specialized transport such as motorcycles having tires with special treads for riding in mud, expensive four-wheel drive vehicles, or even boats (11). In efficacy trials, study participants should understand the need to maintain continuous follow-up, as this will have direct impact on the ability to meet the primary objective.

Informatics With advancing technology in informatics, clinical field sites in developed countries are more commonly using electronic data entry systems whereby study data are entered directly via the World Wide Web. These systems are challenging to implement in developing countries for several reasons, so sponsors should expect to invest considerable financial resources and effort. Since Internet access is not always reliable or even available in many areas of developing countries, there is the logistical challenge of installing and maintaining Internet access. While these connections may be available in urban settings, they are still unusual in most rural settings. Investigators and sponsors may decide to install a satellite connection, but this is also costly and, depending on the environment, may be variably successful. Investigators should work with the sponsor to secure training for key personnel who can perform maintenance and troubleshooting of both hardware and software. Also, local personnel, who likely have varying levels of computer literacy, must be trained on the data entry system; this is increasingly possible as personal digital assistants (PDA) or portable laptop computers are used to collect data in the field (12). A census of over 200,000 persons living in several quartiers in Bamako, Mali was completed using laptop computers in the field to enter data and then uploading the data to a local server at a central site. Even when the costs of Internet access and training have been provided, the issue of sustainability is important. At the end of the trial, funding must be obtained to maintain the equipment left after the trial has been completed.

To avoid this, investigators have to find additional funds from alternate sources.

MULTICENTER CLINICAL TRIALS/TRIAL NETWORKS To study representative populations and to achieve enrollment targets, trials are often conducted at multiple sites. The exchange among investigators, especially in developing countries, offers an opportunity to share experiences and expertise. Often, the challenges confronting investigators are similar across sites, and working together solutions found for problems at one site can be applied at another site. These interactions also promote the formation of investigator networks that can serve as a means to exchange training opportunities and coordinate efforts. As in the case of the AIDS Vaccine Integrated Project, among its many activities to advance the development of a vaccine against Human Immunodeficiency Virus (HIV), investigators have established a network of clinical sites in South Africa (13), and similar networks are being established for malaria vaccine trial sites. These networks are building capacity by establishing harmonized procedures and training clinical and laboratory staff. These activities require a great deal of collaborative effort, which can take much time to harness. Therefore, they should be built into the vaccine development plan so that by the time a vaccine candidate is available for trial, the sites are prepared to implement the protocols.

ISSUES RELATED TO TRIAL DESIGN AND IMPLEMENTATION Investigators must also consider certain issues that impact the design and implementation of the trial. Locally generated data that quantify the burden of the disease of interest are necessary to create awareness and promote community participation as well as to make the necessary sample size calculations for trials. The design of the trial, specifically the choice of end points, may also need to be adjusted in a developing country setting. Promoting community participation and sensitivity to cultural differences impact the informed consent process and the recruitment process of the trial.

DISEASE BURDEN DATA While the potential benefit of a vaccine may be evident to international experts, the most persuasive tool to generate interest is local disease burden data. In fact, one of the limitations to the introduction of vaccines in developing countries has been the lack of these data to demonstrate the need for and, later, the impact of these preventive measures. For example, despite the availability of an effective vaccine against Haemophilus influenzae type b (Hib) infections, vaccine coverage among developing countries is only 42%, partly because of the lack of convincing disease burden and vaccine effectiveness data in Asia (14). When available, these data should be shared with the study population as well as the local decision makers. The introduction of Hib vaccine in Mali was largely based on hospital-based surveillance, which demonstrated a very high burden among infants, particularly those six to seven months where the incidence was 370 cases per 100,000 infant population (15). These data were presented to local government and Ministry of Health officials who, having realized the toll of Hib on their population, worked to seek funding from the Global Alliance for Vaccines and Immunization (GAVI) to introduce

Chapter 4: Special Issues in Performing Vaccine Trials in Developing Countries

the vaccine. In this case, disease burden data directly encouraged involvement of local stakeholders to advocate for vaccine introduction. Whereas the efficacy of Hib vaccine had been demonstrated in the Gambia (16) and its efficacy in Mali was anticipated, the same is not true for rotavirus vaccine. In 2006, SAGE (Strategic Advisory Group of Experts of the World Health Organization) stated that ‘‘much needed efficacy data should be collected in Africa and Asia’’ (17). Then in 2007, GAVI announced it would provide support for the introduction of rotavirus vaccine pending the results of safety and efficacy trials in these areas (18). As a result, efficacy trials of the two currently approved vaccines are presently underway in Africa and Asia. These trials are being conducted at field sites known for their work in enteric disease surveillance as well as expertise in the field of clinical trials. Disease incidence is a critical factor considered in the selection of trial sites. Disease burden data also generate sponsors’ interest, especially for efficacy trials. In this case, local incidence data allow a sample size to be calculated to demonstrate vaccine efficacy; in addition, the existence of such data document that the site has experience in identifying the end point cases of interest (19). In some cases, as part of their vaccine development program, sponsors may sensitize the community and local decision makers by supporting observational studies describing the epidemiology of the target disease. If the candidate vaccine is found to be effective and is subsequently introduced into the routine vaccination program, continued disease surveillance helps to collect impact data, which can influence local decision makers and stakeholders regarding the need to continue funding of this new vaccine.

TRIAL END POINTS Often, the ultimate goal of vaccine development is to decrease the mortality rate associated with that particular infection. In developing countries, many deaths occur at home and investigators frequently have trained personnel to perform verbal autopsies to determine the cause of death. However, these questionnaires have low specificity and it is difficult to ascertain the true cause of death. Consequently, investigators usually measure all-cause mortality especially when the target disease is difficult to diagnose from a verbal autopsy (e.g., malaria) (20). During a large-scale trial of pneumococcal conjugate vaccine in the Gambia (21), investigators found that their ability to measure the impact of the vaccine on deaths due to acute lower respiratory tract infection (ALRI), a tertiary end point, depended on the sensitivity and specificity of the verbal autopsy technique. If deaths that were not due to ALRI were misclassified as such then the power to measure vaccine efficacy on this outcome diminished. As a result, investigators decided to use all-cause mortality as a tertiary end point. This decision had other advantages as pneumococcal vaccine could have an effect on other diseases and this would not be captured in ALRI-specific mortality data. In the end, investigators thought that an all-cause mortality analysis would be more convincing from a public health standpoint (22). In most cases, sample sizes sufficient to support the use of mortality end points are not practical, in which case surrogate end points must be chosen. For example, in malaria vaccine trials, end points that are increasingly distant from malaria mortality have been proposed or used: severe malaria (23), clinical malaria episodes (1), and parasite density (24). The relevance of the end point to the outcome of public health

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interest must be weighted against the practical and cost limitations on the study size.

COMMUNITY PARTICIPATION To ascertain whether a clinical trial can be conducted in a given area, one must engage the local population through community meetings to increase awareness and to gauge willingness to participate in trials. In some cultures, these meetings may be a requirement before investigators may begin recruitment for any research activities. Community outreach activities can be conducted through the use of local media and meetings. For their large-scale pneumococcal efficacy trial, investigators in the Gambia communicated via radio, public performances, and community meetings. These venues were used to publicize the trial as well as to educate the community regarding the target disease (11). Local beliefs regarding the cause of a particular disease should be understood as these can have an impact on treatment seeking (25) as well as willingness to participate in prevention trials. Even when a community is sensitized to a particular disease, this knowledge may not necessarily indicate that they are willing to participate in research. Motivation may be affected by a number of factors such as previous individual and community experiences with research and investigational products, attitudes toward vaccines and western medicine, understanding of the etiology of disease, and concerns regarding confidentiality. Investigators can assess the importance of these factors by conducting meetings with the various local leaders and the community at large. Through meetings, investigators and the community can exchange views regarding the positive and negative aspects of previous studies and expectations for future studies. In the case of HIV vaccine trials, investigators have been conducting community outreach activities before the implementation of trials; focus groups are interviewed on their concerns regarding participation in these trials. Investigators have found that persons realize that HIV has taken a tremendous toll on their community and so are motivated to participate, but are concerned about receiving an experimental vaccine as well as possible stigmas associated with participation (26–28). With this information, activities addressing these particular concerns can be designed. In some cultures, community meetings must be held to obtain community agreement or consent to participate in a trial, that is, recruitment cannot begin until the community agrees to have the research take place. In Mali, in keeping with social norms, a series of community meetings is held with local leaders and community representatives, offering multiple opportunities for the population and investigators to interact (29,30). These meetings are attended by the local chief as well as their advisors, including the leaders among the women and youth of the community. Local government officials may also attend. Involvement of local health care providers, particularly traditional healers, is also important because these individuals are usually very influential in medical decision making. By combining the science and public understanding of the disease, investigators can engage the community so that they become invested in the research.

WOMEN’S PARTICIPATION IN RESEARCH Research in women can be challenging for a variety of reasons and has been well described in the area of HIV vaccine research (31). In many cultures, women lack autonomy in medical

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decision making and may not be permitted to provide their individual consent. Moreover, the informed consent process may be challenged by the low literacy rates found among women in developing countries. Recruitment of married women also provides challenges as the protocol may require pregnancy testing or the agreement of the woman to adhere to family planning techniques. These procedures may be objectionable for either religious or cultural reasons and could threaten her position in the family. Despite the many obstacles, through the implementation of awareness programs aimed at men and women, the community can be sensitized and women can accept to participate.

CULTURAL DIFFERENCES The challenges of working in the context of different cultures are considerable. Literacy rates in developing countries are relatively low, which impacts the informed consent process. Consent materials may need to be translated into local languages and visual and auditory techniques may need to be used to impart the necessary information. A clinical trial site in Mali has consent forms translated and recorded by the national translation service into local languages so that participants can listen to rather than read the content. Concepts such as randomization must be explained using locally relevant examples such as the likelihood of picking a particular peanut from a sack full of peanuts. In pediatric trials, obtaining the appropriate signature on a consent form may also be difficult as children are often charged to family members while the natural parents are living elsewhere. Children may be ‘‘adopted’’ by distant family without seeking legal documentation from the local authorities. Multiple generations may share a single household, resulting in grandparents being considered as the primary decision makers. All of these particularities challenge the informed consent process compared with how it is conducted in industrialized countries, and sponsors often have to remain open-minded about what is acceptable. Often, trials of vaccines already licensed for use in industrialized countries are performed under markedly different epidemiological and demographic conditions in developing to establish local efficacy. In this case, sponsors may wish to apply in the developing country venue the procedures and techniques that were successful elsewhere; this approach is variably successful. For example, whereas post-vaccination daily temperature monitoring by participants may be common practice in an industrialized or transitional country, in a poor developing country most people may not be accustomed or able to collect this information. Moreover, sponsors may request that case identification be made at organized health facilities because there can be greater certainty regarding the clinical findings. In contrast, locals may prefer to visit traditional healers because of lower cost, trustworthiness, or greater convenience. In this case, sponsors must rely on the local investigators’ experience, and as a result, case identification methods between industrialized and developing country settings may differ.

SUSTAINABILITY OF STUDY-RELATED ACTIVITIES/STANDARD OF CARE Populations that participate in research often benefit from the strengthening of local health services that occur as a byproduct in the area where the trial was conducted. In many cases, health

care personnel are recruited, clinics refurbished, disease surveillance implemented, and participants educated in health matters. Since vaccine trials can last several years, the population becomes accustomed to this standard of care. When study activities have not been incorporated into routine health services, study participants come to rely on care from the study personnel and their utilization of the public health care system can be affected. As a result, it is important to discuss these issues with the community and the sponsor so that study material may be donated to the community at the end of the trial. In addition, local personnel, including governmental health care providers, should be used whenever possible so that these newly trained and sensitized personnel can remain at their usual post after the trial, thereby building sustainability. Thus, the community continues to benefit long after the trial has ended.

ISSUES RELATED TO TRIAL OVERSIGHT As with any trial, there is a considerable amount of oversight from external groups, including ethical review committees and data and safety monitoring boards. Such oversight groups have certain challenges ahead of them that are specific to the developing country setting.

ETHICAL REVIEW COMMITTEES AND INSTITUTIONAL REVIEW BOARDS In most cases, international vaccine trials are reviewed by local ethical review committees in the host country and external institutional review boards (IRBs) in the sponsor’s country and in any collaborating countries. The ethical issues surrounding protocol review by multiple committees are addressed elsewhere; logistical aspects will be discussed here. Since relatively few vaccine clinical trials have been conducted in most developing countries, ethical review committees have variable levels of experience and expertise. To demonstrate that they are following international norms, committees obtain an assurance such as the Federalwide Assurance (FWA), which is provided by the U.S. Department of Health and Human Services to indicate that the committee is in compliance with Code of Federal Regulations Title 45 Part 46. However, committees may not be acquainted with all of their assigned duties and sponsors, and investigators may find themselves requesting additional documentation or submitting unrequested annual reports. These requests may go beyond what the committee expects and may exceed the committee’s resources. Nonetheless, these interactions should be considered part of capacity building as the committees gain expertise in the review of clinical vaccine trials. Committees in developing countries may have a limited support staff, resulting in long turnaround times for review. In some cases, ad hoc committees may be convened, making subsequent reviews of protocols problematic, as the committee has to reconvene. Reviews by each collaborating institution can be difficult to coordinate, especially when they make conflicting requests. Nonetheless, any local requests regarding the study design must be agreed on by all participating institutions so that results may be interpretable across sites. Obtaining serial approvals (awaiting final approval from one committee before submitting for initial review by another committee) can take months or even years. To avoid long delays in obtaining final ethical approval, it is often most efficient to submit the protocol to all committees simultaneously, incorporate all requested

Chapter 4: Special Issues in Performing Vaccine Trials in Developing Countries

changes into a harmonized protocol, and then get final approval on a second or third revision. Typically, one or more committees offer provisional approval of the first version, making it possible to incorporate changes requested by another committee into an amended protocol, which can often be approved more quickly. Some U.S. IRBs insist on having the protocol approved by the local IRB prior to their own review. Because of the long delays that can result from such an approach, it is advisable to negotiate this point or even to submit a protocol for review without having met this condition and allow the review to proceed with the expectation of a request for the missing approval, which may be in hand by the time other IRB concerns are addressed. Conflicts between IRBs are discussed in detail in the chapter entitled ‘‘Ethical Considerations in the Conduct of Vaccine Trials in Developing Countries.’’

DATA SAFETY MONITORING BOARDS Data safety monitoring boards (DSMBs) are convened to review safety data collected during the trial. DSMBs for vaccine trials may comprise experts in infectious disease, a biostatistician, and a bioethicist. Further, at least one of the experts should be from a developing country and the bioethicist should have experience in research in similar settings (32). While it may be difficult to identify members from the local scientific community, the inclusion of a local expert on the board is valuable, even critical, as they may be able to elucidate any unexpected findings among the local study population. Moreover, the inclusion of local individuals in an international committee of this expertise builds capacity as they become acquainted with clinical trials and may serve on future DSMBs. Since local investigators may have a limited experience in reporting to a DSMB, the presence of such a committee also increases their capacity and promotes sustainability by encouraging proper reporting of adverse events. The board may offer guidance on the adverse event reporting system as well as the formatting of summary reports for their review. While not usually one of their roles, one DSMB found that the educational component of their interactions with local investigators became their legacy as the conduct and quality of future studies was likely to improve as a consequence of these activities (32).

CONCLUSION The rewards of conducting vaccine trials in developing countries are considerable, and include the benefit of collecting important data regarding the safety and efficacy of essential vaccines, as well as the development of capacity of trial sites so that future research can be conducted and other vaccines developed. Clinical vaccine trials afford developing country investigators the opportunity to improve their skills while taking advantage of the support of sponsors and the greater international research network; each party has a stake in supporting sustainable research. As local investigators perform more trials with financial support from sponsors, infrastructure is developed so that clinical and laboratory personnel are trained and the facilities are updated to meet study requirements. Moreover, membership in clinical trial networks or international collaborations and experience with study monitoring further enhances the learning experience for investigators as they share and exchange information with their regional

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colleagues and international experts. In turn, investigators educate the international community on the particularities of successfully conducting research in their region. Ultimately, the greatest reward belongs to the local community; they are able to take advantage of potentially life-saving vaccines sooner rather than later.

REFERENCES 1. Alonso PL, Sacarlal J, Aponte JJ, et al. Efficacy of the RTS,S/ AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomised controlled trial. Lancet 2004; 364:1411–1420. 2. Deen JL, Clemens JD. Issues in the design and implementation of vaccine trials in less developed countries. Nat Rev Drug Discov 2006; 5:932–940. 3. Glass RI, Bresee JS, Turcios R, et al. Rotavirus vaccines: targeting the developing world. J Infect Dis 2005; 192(suppl 1):S160–S166. 4. Gotuzzo E, Butron B, Seas C, et al. Safety, immunogenicity, and excretion pattern of single-dose live oral cholera vaccine CVD 103HgR in Peruvian adults of high and low socioeconomic levels. Infect Immun 1993; 61:3994–3997. 5. Richie EE, Punjabi NH, Sidharta YY, et al. Efficacy trial of singledose live oral cholera vaccine CVD 103-HgR in North Jakarta, Indonesia, a cholera-endemic area. Vaccine 2000; 18:2399–2410. 6. Laforce FM, Konde K, Viviani S, et al. The meningitis vaccine project. Vaccine 2007; 25(suppl 1):A97–A100 [Epub May 7, 2007]. 7. Kshirsagar N, Mur N, Thatte U, et al. Safety, immunogenicity, and antibody persistence of a new meningococcal group A conjugate vaccine in healthy Indian adults. Vaccine 2007; 25(suppl 1):A101–A107. 8. McCarthy M. Project seeks to ‘‘fast track’’ rotavirus vaccine. Lancet 2003; 361:582. 9. Cunliffe N, Nakagomi O. Introduction of rotavirus vaccines in developing countries: remaining challenges. Ann Trop Paediatr 2007; 27:157–167. 10. Sulo J, Chimpeni P, Hatcher J, et al. Chlorproguanil-dapsone versus sulfadoxine-pyrimethamine for sequential episodes of uncomplicated falciparum malaria in Kenya and Malawi: a randomised clinical trial. Lancet 2002; 360:1136–1143. 11. Cutts FT, Enwere G, Zaman SM, et al. Operational challenges in large clinical trials: examples and lessons learned from the Gambia pneumococcal vaccine trial. PLoS Clin Trials 2006; 1:e16. 12. Missinou MA, Olola CH, Issifou S, et al. Short report: piloting paperless data entry for clinical research in Africa. Am J Trop Med Hyg 2005; 72:301–303. 13. Ferrantelli F, Butto S, Cafaro A, et al. Building collaborative networks for HIV/AIDS vaccine development: the AVIP experience. Springer Semin Immunopathol 2006; 28:289–301. 14. Morris SK, Moss WJ, Halsey N. Haemophilus influenzae type b conjugate vaccine use and effectiveness. Lancet Infect Dis 2008; 8:435–443. 15. Sow SO, Diallo S, Campbell JD, et al. Burden of invasive disease caused by Haemophilus influenzae type b in Bamako, Mali: impetus for routine infant immunization with conjugate vaccine. Pediatr Infect Dis J 2005; 24:533–537. 16. Mulholland K, Hilton S, Adegbola R, et al. Randomised trial of Haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in Gambian infants. Lancet 1997; 349:1191–1197. 17. Conclusions and recommendations from the Immunization Strategic Advisory Group. Wkly Epidemiol Rec 2006; 81:2–11. 18. New and underused vaccines support (NVS). Available at: http://www.gavialliance.org/support/what/nvs/index.php. Accessed October 3, 2008. 19. Adegbola RA, Mulholland EK, Falade AG, et al. Haemophilus influenzae type b disease in the western region of The Gambia: background surveillance for a vaccine efficacy trial. Ann Trop Paediatr 1996; 16:103–111.

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20. Guinovart C, Alonso PL. Methods for determining vaccine efficacy and effectiveness and the main barriers to developing a fully deployable malaria vaccine. Am J Trop Med Hyg 2007; 77:276–281. 21. Cutts FT, Zaman SM, Enwere G, et al. Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial. Lancet 2005; 365:1139–1146. 22. Jaffar S, Leach A, Smith PG, et al. Effects of misclassification of causes of death on the power of a trial to assess the efficacy of a pneumococcal conjugate vaccine in The Gambia. Int J Epidemiol 2003; 32:430–436. 23. Lyke KE, Dicko A, Kone A, et al. Incidence of severe Plasmodium falciparum malaria as a primary endpoint for vaccine efficacy trials in Bandiagara, Mali. Vaccine 2004; 22:3169–3174. 24. Genton B, Betuela I, Felger I, et al. A recombinant blood-stage malaria vaccine reduces Plasmodium falciparum density and exerts selective pressure on parasite populations in a phase 1-2b trial in Papua New Guinea. J Infect Dis 2002; 185:820–827. 25. Ofovwe GE, Ibadin OM, Ofovwe EC, et al. Home management of febrile convulsion in an African population: a comparison of urban and rural mothers’ knowledge attitude and practice. J Neurol Sci 2002; 200:49–52.

26. Olin J, Kokolamami J, Lepira FB, et al. Community preparedness for HIV vaccine trials in the Democratic Republic of Congo. Cult Health Sex 2006; 8:529–544. 27. Nyamathi AM, Suhadev M, Swaminathan S, et al. Perceptions of a community sample about participation in future HIV vaccine trials in south India. AIDS Behav 2007; 11:619–627. 28. Suhadev M, Nyamathi AM, Swaminathan S, et al. A pilot study on willingness to participate in future preventive HIV vaccine trials. Indian J Med Res 2006; 124:631–640. 29. Diallo DA, Doumbo OK, Plowe CV, et al. Community permission for medical research in developing countries. Clin Infect Dis 2005; 41:255–259. 30. Roberts L. Infectious disease. Clinical trials: dispelling suspicions, building trust in Mali. Science 2008; 320:1714. 31. Mills E, Nixon S, Singh S, et al. Enrolling women into HIV preventive vaccine trials: an ethical imperative but a logistical challenge. PLoS Med 2006; 3:e94. 32. Chen-Mok M, VanRaden MJ, Higgs ES, et al. Experiences and challenges in data monitoring for clinical trials within an international tropical disease research network. Clin Trials 2006; 3:469–477.

5 Long-Term Evaluation of Vaccine Performance: Methodological Issues for Phase III and Phase IV Studies John D. Clemens International Vaccine Institute, Seoul, Korea

Abdollah Naficy Parasitology and International Programs Branch, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

Malla R. Rao International Tropical Diseases Research/Epidemiological Studies, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, U.S.A.

Hye-won Koo Pharmaco-Economics and Health Outcomes Research, Medical Department, GlaxoSmithKline Korea, Seoul, Korea

INTRODUCTION Rigorous and meticulous scientific evaluations are required before the introduction of a vaccine into public health practice. In this chapter, we provide an overview of several methodological considerations that are relevant to field evaluations of the clinical protection conferred by vaccine candidates that have proved suitably safe and immunogenic in earlier animal and human studies.

TYPES OF EVALUATIONS Evaluations of the protection conferred by vaccines against clinical infections occur both before and after vaccine licensure. Before licensure, vaccines are conventionally tested in an orderly sequence of ‘‘phases.’’ In the U.S. Code of Federal Regulations, the phases of clinical trials are described using Arabic numerals (phase 1, phase 2, phase 3), whereas in World Health Organization publications, Roman numeration is generally used (e.g., phase I, phase II, phase III). Because of the international dissemination of this book, in this chapter, we will utilize the latter. Phase I and II studies evaluate the safety, immunogenicity, and transmissibility of vaccine candidates in relatively small numbers of subjects. For the relatively few diseases in which volunteer challenge models have been established, such as cholera, malaria, influenza, and shigellosis, phase II studies (sometimes called phase IIb studies) may also evaluate the protection conferred by vaccination against an experimental challenge of volunteers with the target pathogen. Confusingly, the term phase IIb is also used for trials in which relatively small samples are enrolled to gain preliminary estimates of protection against naturally occurring disease (1). For vaccines found to perform suitably well in phase I and II

studies, phase III studies are conducted to provide rigorous evidence about vaccine safety and efficacy. Phase III studies are constructed as experiments with clear hypotheses and are conducted in a population that normally experiences the disease against which the vaccine is targeted. After licensure, clinical protection may be monitored in several ways with phase IV studies. Sometimes protection is assessed by evaluating postimmunization immune responses to vaccines given in routine practice. When such studies are conducted in an area endemic for the target infection for the vaccine, it is necessary to evaluate immune responses in both vaccinees and nonvaccinees so that immune responses attributable to vaccination can be distinguished from responses to natural infections. Examples of such studies include measurement of serum hemagglutination inhibition antibodies after measles vaccination and assessments of cutaneous delayed hypersensitivity to tuberculin after bacille Calmette-Gue´rin (BCG) vaccination (2,3). Such studies may provide useful information about the immunogenicity of vaccines, and for vaccines for which immunological correlates of clinical protection are well established, they can serve as appropriate methods for monitoring the protection conferred by vaccines that have been deployed in public health practice. However, because immunological correlates of protection are not known for many vaccines, direct clinical assessments of vaccines versus nonvaccinees are often required to determine whether a vaccine, as routinely administered in public health practice, is suitably safe and protective. Recent examples of such postmarketing surveillance studies include assessments of the clinical effectiveness of Haemophilus influenzae type b (Hib) polysaccharide vaccine in children (4) and of pneumococcal polysaccharide vaccine in the subgroups of individuals who are

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at high risk for serious pneumococcal disease (5). Such studies are usually designed in an observational rather than an experimental fashion, often as cohort or case-control studies (6), although there has been recent interest in evaluating licensed vaccines in realistic, public health contexts with use of experimental designs (further described in the section ‘‘Clarification of the Perspective of the Trial’’) (7). In this chapter, we focus primarily on the use of randomized clinical trials (RCTs) for phase III vaccine evaluations. We also briefly discuss approaches to evaluating the vaccine protection and safety in phase IV studies.

RANDOMIZED CLINICAL TRIALS OF VACCINES Overview The RCT has become recognized as the most powerful research design for providing scientifically credible evidence about therapeutic efficacy (8,9), leading many national drug regulatory agencies to require evidence of efficacy from properly conducted RCTs before licensure of a new vaccine. Indeed, it may be anticipated that most, if not all, field evaluations of efficacy of experimental vaccines conducted in the future will be designed to conform to the RCT paradigm. Figure 1 provides a diagrammatic outline of a simple, two-group RCT of an experimental vaccine. Individuals recruited from a target population are enrolled for the study after acquisition of informed consent and ascertainment of eligibility. Study participants are then randomized to the experimental vaccine group or to a comparison (control) group. After randomization, study participants are followed concurrently to detect target infections with onsets during a defined period of follow-up. The incidence of the target infection in the different groups is then compared to assess whether or not protection occurred in the group receiving the experimental vaccine, relative to the comparison group. Incidence may be expressed as a rate, the number of detected infections divided by the cumulative person-time at risk; or as a risk, the number of infected persons detected over some specified period of time divided by the number of persons at risk. If we take group B as the comparison group, such contrasts are typically expressed as rate ratios or risk ratios (RRs), both based on the occurrence in group A divided by that in group B. The conventional index of protection, protective efficacy (PE), is then calculated as PE = (1
RR)  100% (10). PE reflects the proportionate reduction of the incidence of the

target infection in the experimental vaccine group relative to the comparison group. A value of 0% denotes no protection, that of 100% corresponds to complete protection, and negative values indicate a lower incidence in the comparison group than that in the experimental vaccine group.

Target Population In any trial, it is first necessary to choose an appropriate target population for the evaluation. Clearly, it will be necessary to select a population in which the target infection occurs predictably and with a sufficient frequency to evaluate whether vaccination reduces the incidence of the target infection. Such populations may be defined on the basis of geographic location as well as age, gender, and a variety of additional characteristics. For example, a classic trial of plasma-derived hepatitis B vaccine was conducted in homosexual men who resided in New York (11), and trials of newer-generation vaccines against leprosy have focused on family contacts of known patients with leprosy (12). Regardless of the target population chosen for the trial, it is necessary to enumerate and characterize the population before the trial to enable later assessment of whether or not the final group for the study was representative of the intended target population.

Recruitment After defining the target population for study, investigators must next recruit participants from this population. An important element of recruitment is informed consent. The particular requirements for obtaining informed consent may vary according to the age of the subject and the subject’s legal capacity to give informed consent as well as the particular milieu for the trial. In general, acquisition of informed consent, which should be documented in writing, indicates that subjects have agreed to participate after understanding the purposes and elements of the trial as well as the possible benefits and risks of participation and after being guaranteed that their decisions to participate and to continue participating are completely voluntary (13).

Eligibility Consenting individuals must usually fulfill additional eligibility criteria before enrollment in a trial. At a minimum, these eligibility criteria should exclude any persons with absolute

Figure 1 A schematic of the sequence of events in a two-group, randomized, controlled vaccine trial. In this sequence, the study population is assembled from a target population and is then randomized to receive an experimental vaccine or a comparison agent. The experimental and comparison groups are then followed longitudinally and concurrently to detect target infections.

Chapter 5: Long-Term Evaluation of Vaccine Performance

indications for, or contraindications to, any of the agents under evaluation (14). Such exclusions are ethically necessary if the trial is to use an impartial allocation procedure, such as randomization, that assigns compared agents without reference to the individual needs or desires of the participants. Additional eligibility criteria may be imposed to further restrict the characteristics of the population under evaluation. Persons with serious underlying illnesses, who may not respond to vaccination, may be excluded in some trials. Investigators may also choose to focus on immunologically naive individuals who have not previously experienced the target infection and have not been immunized before with a vaccine against this infection. A trial may also limit participants to persons who are at a very high risk of acquiring the target infection by virtue of a history of exposure or of relevant host characteristics. Although such restrictions are commonly imposed to increase the detectability of vaccine protection, they may also substantially modify the research question that the trial addresses.

Allocation and Administration Depending on the purpose of the trial, a group receiving an experimental vaccine may be compared with a group assigned to a different vaccine against the target infection, to an agent not anticipated to affect the risk of the target infection, or to no agent at all (15). Postponement of allocation of the compared agents until after acquisition of informed consent and ascertainment of eligibility helps to ensure that decisions about recruitment, participation, and eligibility do not depend on which of the agents has been assigned (16,17). Moreover, because this sequence ensures that only enrolled subjects are assigned to the compared agents, it minimizes irregularities created by persons who drop out of the study after assignment but before receipt of an agent under study. A powerful mechanism to prevent such biases in the intake of participants is to conceal the identities of the compared agents from both participants and investigators (double blinding) (18). Double-blinded administration of agents also safeguards against a bias that can occur if participants are given the opportunity to choose additional measures to prevent the target infection on the basis of knowledge about which of the compared agents has been received. It is important that the compared agents be allocated with a nondiscretionary method that, as far as possible, creates groups with equivalent baseline risks for the target infection under evaluation. The most powerful technique for accomplishing this goal is to allocate eligible subjects to different agents in a formally randomized fashion (19–21). Formally randomized allocation, a prerequisite for modern trials of new vaccines, has the additional desirable property of providing a theoretical basis for statistical appraisals of differences in the occurrence of outcome events as well as other characteristics in the compared groups (22).

Surveillance Vaccine field trials typically conduct surveillance for at least four types of events. First, systematic surveillance must be instituted for the target infection that the vaccine is intended to prevent. Arrangements must be made for systematic collection of data relevant to the diagnosis of the target infection, and the study must be designed to ensure that members of the compared groups have an equivalent probability of receiving

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relevant diagnostic procedures when they develop the target infection (18). It is also mandatory that the diagnostic evidence be interpreted in an objective manner that is not influenced by knowledge of which agent has been received. Double-blinded surveillance constitutes the most powerful method to safeguard against biased detection and ascertainment of outcomes, although alternative strategies are available when double blinding is not possible (23). A second important goal of surveillance is to detect adverse reactions after vaccination. Preliminary studies before the trial will usually have identified likely candidate side effects as well as the time frame for the development of these side effects, which is usually a period of a few days after vaccination. Participants should be monitored for these anticipated events as well as for the possibility of less likely reactions that may occur with longer latency periods after vaccination. Immune responses to vaccination constitute a third category of events to be monitored. Evidence of expected levels of immune responses to vaccination documents that the vaccine lots under evaluation were properly prepared, stored, and administered. If a vaccine proves effective in preventing the target disease, the trial may permit assessment of the relationship between the levels of response and the degree of protection. A fourth category of events under surveillance, competing events, are outcomes other than occurrence of the target infection that terminate a participant’s period of follow-up (16,17). Follow-up arbitrarily stops at the end of a study but may also be prematurely terminated by loss from the study for such reasons as death, refusal to continue participation, and migration away from the study area. Such events are important for several reasons. Deaths, in addition to terminating followup, may themselves be important outcome events. Moreover, all of these events create the opportunity for unequal periods of follow-up among participants and therefore must be considered in calculating periods at risk for expressing the incidence of the target infection. Finally, if the tendency to become lost to follow-up differs in the compared groups, the losses themselves may create a differential opportunity to detect the target infection in the groups and may thereby bias estimates of the comparative occurrence of the target infection. As with surveillance for the target infection, suitably objective methods, such as double blinding, are necessary to prevent biased detection and ascertainment of side effects, immune responses, and competing events.

ADDITIONAL ISSUES FOR PHASE III RANDOMIZED CLINICAL TRIALS OF VACCINES Although the basic paradigm of a controlled vaccine field trial may seem reasonably straightforward, additional issues require consideration in both the design and the analysis of a vaccine trial. In the following sections we outline several of these issues.

Posing Research Questions for the Trial Posing the research questions for the trial appropriately and with adequate specificity is crucial to the success of a phase III trial, since research questions guide the design, conduct, and analysis of the trial. Modern vaccine trials are designed to address primary research questions, evidence bearing on which will be considered by regulatory agencies in deliberations about vaccine licensure, and secondary questions, which address additional scientific issues of interest. Adequate

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formulation of the primary research question(s) demands clear specification of the target population for the study; the formulation and constituents of the vaccine together with its mode and schedule of administration; the comparison agent to be used as a control agent; the target infections whose occurrence will serve as the basis for estimating vaccine protection; the adverse event outcomes whose occurrence will serve as the basis for evaluating vaccine safety; and the immune outcomes whose occurrence will serve as a basis for assessing vaccine immunogenicity. Each of these features needs to be articulated in a way that accurately reflects how a vaccine will be used in practice, to ensure that a trial’s results will meet the regulatory expectations for vaccine licensure.

Demarcating the Type of Vaccine Protection to Be Measured When we administer a vaccine to a subject, our hope is that the vaccine will elicit relevant forms of immunity that will make that subject less susceptible to becoming ill when exposed to the target pathogen. This protection, which reflects the effects of the vaccine on the individual independently of whether or not other individuals in the same population are also vaccinated, is called direct protection (24). However, for most vaccines, we attempt to immunize populations rather than individuals. For pathogens that are transmitted from person to person, targeting a population rather than an individual for vaccination may have the added effect of reducing the intensity of transmission of the pathogen in the population. One possible result is that indirect protection of nonimmunized, susceptible individuals may occur, and they may be protected because they are less likely to come into contact with the pathogen (24,25). An additional possible result is that the protection of vaccinees may be augmented, since the protection conferred by many vaccines is greater against a small challenge inoculum of a pathogen than that against a heavy challenge inoculum. This augmented protection of vaccinees is termed total protection. Depending on its design, a phase III trial of a vaccine can measure either direct protection or total protection of vaccinees. To help measure the former, allocation of subjects to vaccine and the comparison agents must not create high concentrations of vaccinees within the geographical units within which the pathogen is transmitted. This is usually accomplished by randomizing individuals rather than groups and by randomizing within blocks that balance the numbers of vaccinees and controls within epidemiologically relevant groups of individuals. To permit estimation of total protection of vaccinees, as well as indirect protection of nonvaccinees, a different strategy is typically used: clusters of individuals rather than individuals per se serve as the units of allocation (26). If we imagine a trial in which clusters are randomly allocated either to a vaccine under study or to an agent with no activity against the target pathogen (e.g., a placebo) and if we assume that not all members of a cluster will actually receive the assigned agent, we can see that several subgroups of subjects are formed (Fig. 2): vaccinated and nonvaccinated subjects within the clusters allocated to the vaccine, and recipients of the control agent and nonrecipients of the control agent within the clusters allocated to receive the control agent. Comparison of the incidence of the target infection in vaccinees versus that in recipients of the control agent estimates total protection. Comparison of nonrecipients of vaccine in the vaccinated clusters with nonrecipients of the control agent in control clusters allows estimation

Figure 2 A diagram of measurement of vaccine protection in a cluster-randomized vaccine trial. Two hypothetical clusters are shown, and the types of individuals to be contrasted for measurement of different types of vaccine protection are identified.

of indirect protection. Finally, overall comparison of the incidence of infection in clusters allocated to the vaccine versus clusters allocated to the control agent, regardless of whether an agent was actually received, permits estimation of the overall protection of vaccination in the entire study population targeted for the vaccine (27). While cluster-randomized trials offer the most straightforward experimental designs for measuring these different types of vaccine protection, a recently published reanalysis of a trial of killed oral cholera vaccines in Bangladesh demonstrated a new method by which geographic mapping of the trial population can assist in evaluating both total and indirect vaccine protection even within an individually randomized trial. With this method, suitable geographic clusters are defined, and vaccine coverage is correlated with incidence of the outcome infection among the clusters. An inverse relation between coverage and outcome incidence among vaccinees would denote total vaccine protection and that among nonvaccinees would correspond to indirect protection (28). Traditionally, regulatory agencies have required that phase III trials be individually randomized, owing to an assumption that such designs assure estimation of direct protection per se and to a preference that trials measure the ‘‘intrinsic’’ protection of the vaccinated individual, irrespective of the vaccine coverage of other persons in contact with that individual. However, as illustrated by the Bangladesh cholera vaccine trial, described earlier, even individually randomized trials may yield measures of vaccine protection that are not purely direct. A hint that the trial designs required by regulatory agencies may be evolving comes from a recent phase III trial of pneumococcal polysaccharide-protein conjugate vaccine in native American infants in the United States, which employed cluster randomization and was accepted as a pivotal trial for registration of the vaccine in the United States (29).

Clarification of the Perspective of the Trial To ensure scientific credibility of the results, vaccine trials must be designed to safeguard against bias. However, even after a trial has been suitably designed to prevent bias, an additional

Chapter 5: Long-Term Evaluation of Vaccine Performance

fundamental decision remains: Should the trial be designed to measure the intrinsic effects of vaccination when it is given under ideal conditions, or should the design ensure that the results can be used to predict how well the vaccine will perform in public health practice? Trials designed to accomplish the former goal are often termed efficacy trials, and trials constructed to accomplish the latter objective are frequently referred to as effectiveness trials (26,30). Each type of trial has different requirements for the selection of study subjects, choice of the compared agents, units of allocation, selection and definition of outcome events, and demarcation of the participants to be analyzed (26,31–33). An efficacy trial may attempt to restrict study subjects to persons who are likely to respond to vaccination and are at such a high risk for the target infection that protection will be readily detectable. For example, early studies of pneumococcal polysaccharide vaccine examined vaccine efficacy in otherwise healthy South African gold miners, who were documented to be at a very high risk of serious pneumococcal infections (34). On the other hand, an effectiveness trial would study groups that are anticipated to be target groups for vaccination after licensure. For pneumococcal vaccine utilized in developed countries, the target group comprises the elderly and others at a high risk because of the underlying disorders (35). That the results of these two selection strategies are not necessarily interchangeable is illustrated by the controversy that arose when several postmarketing surveillance studies suggested that the high levels of efficacy of pneumococcal vaccine observed in healthy individuals were not exhibited by the debilitated groups targeted for public health application (36). To enhance the detectability of vaccine protection, an efficacy trial tests vaccines whose formulations and regimens are designed to yield optimal protection and whose proper administration is assured by idealized experimental conditions. If ethically permissible, an efficacy trial often will contrast the group that receives the new vaccine with a comparison group that receives an agent not anticipated to protect against the target infection. In contrast, an effectiveness trial will test vaccines whose formulations and regimens are most suitable for application in public health practice, will conduct the evaluation in the realistic environment of a public health program, and may, where appropriate, will compare the performance of a new vaccine with that of a standard vaccine directed toward the same disease. Past evaluations of Ty21a oral vaccine against typhoid fever illustrate these differences. An early efficacy trial conducted in Alexandria, Egypt evaluated a preparation of high potency but with little applicability to public health practice because of its inconvenient formulation (37). After demonstration of high efficacy in this early trial, later trials, conducted in Chile, evaluated more convenient enteric-coated capsules and reconstituted lyophilized ‘‘liquid’’ formulations (38–40). Whereas the early trials of this vaccine were placebo controlled, a later trial, designed to answer practical questions relevant to public health implementation, compared only different regimens of the active vaccine (39,41). To measure the intrinsic effects of vaccination, efficacy trials typically seek to measure only direct vaccine protection, since herd protective effects are not intrinsic to the vaccine but depend on the pattern and extent of vaccine coverage of a population. Accordingly, the unit of allocation in an efficacy trial is typically an individual, rather than a cluster of individuals, and additional measures, such as blocked randomization,

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are often used to balance vaccines and nonvaccinees within the groups in which transmission occurs. For live vaccines that can be excreted and transmitted to contacts, such as the Sabin oral polio vaccine, efforts are made to physically isolate vaccinees from controls to prevent ‘‘contamination’’ of the control group by inadvertent vaccination. In contrast, the pragmatic goals of an effectiveness trial are often best met by allocation of clusters rather than individuals, since the goal is to capture the overall benefit of vaccinating a target population and since these benefits include direct as well as herd protection of both vaccinees and nonvaccinees residing in the target population. Trials of capsular polysaccharide–tetanus toxoid (PRP-T) conjugate vaccine against Hib in Chile (42) and England (43) illustrate the use of cluster allocation in effectiveness trials. In the former, community clinics in metropolitan Santiago were randomized either to receive this vaccine mixed with diphtheriatetanus-pertussis (DTP) vaccine at two, four, and six months of age or to receive only DTP. In the latter, districts near Oxford were allocated to receive or not to receive PRP-T in conjunction with DTP at two, three, and four months of age. In both studies, all infants in the groups allocated to vaccine or no vaccine were followed for the occurrence of invasive Hib disease regardless of whether they had received PRP-T. The definition of the target infection for the outcome of an efficacy trial aims to demarcate biological events that are anticipated to be responsive to vaccine-induced immunity even if they may be too narrow in scope to permit judgments about the usefulness of the vaccine in public health practice. For example, it was anticipated form North American volunteer challenge studies that the primary effect of ingestion of an oral B subunit–killed whole-cell cholera vaccine would be to reduce the occurrence of clinically severe cholera (44). An efficacy trial of this vaccine in Bangladesh was therefore designed with the primary goal of assessing vaccine efficacy against clinically severe infections (45). Although prevention of life-threatening cholera is certainly an important public health goal, an effectiveness study might be concerned with the overall impact of vaccination against all diarrheal episodes associated with Vibrio cholerae 01 as well as the effect of vaccination on all episodes of clinically severe diarrhea and on diarrheal deaths. Analyses of efficacy trials, sometimes referred to as per protocol analyses, commonly consider only participants who have received a complete course of a properly administered agent. Among these analyzed participants, evaluations of such trials commonly restrict target infections to events that begin after a window of time during which it is anticipated that participants will have had an opportunity to manifest fully developed immune responses to vaccination (‘‘immunogenic window’’). To minimize the possibility of bias caused by such temporal restrictions, they must be applied equally to each of the compared groups (33). In contrast, effectiveness trials, which are concerned with the effects of a policy to administer a vaccine, would analyze all participants originally randomized to the compared agents and would include all events occurring from the time at which dosing with the vaccine and control agents began. If groups constituted the units of allocation, all targeted members of the allocated groups would constitute the most relevant denominators. Although such an ‘‘intent-totreat’’ analytical strategy can be justified on the basis of the effectiveness perspective of a trial, it has also been argued that this strategy for defining outcomes and populations at risk is necessary to avoid bias from distortion of the original groups created by randomized allocation (46).

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The restricted populations, idealized regimens, responsive outcomes, and analytical strategies of efficacy trials will almost always reduce the sample size requirements of such studies and may also reduce associated costs and logistical demands. An efficacy trial thus constitutes a logical first step in the field evaluation of the protection conferred by a new vaccine (47). Such efficacy trials typically form the basis for regulatory decisions about vaccine licensure. However, if a vaccine yields promising results in an efficacy study but doubts still remain about its practical utility for public health practice, it may then be appropriate to mount one or more effectiveness trials before making decisions about the application of the vaccine in public health programs. Because of this complementarity of efficacy and effectiveness trials, it has been suggested that efficacy trials be termed ‘‘phase IIIA’’ trials and effectiveness trials be termed ‘‘phase IIIB’’ trials (26).

Sample Size Estimation and Interpretation of Background Information Methods for calculating sample sizes required for RCTs are described in detail elsewhere (48). One formula (49) commonly employed for two-group trials in which equal-sized groups are desired gives the size per group (N) as

N¼

ðZa þ Zb Þ2 ½P1 ð1
P1 Þ þ P2 ð1
P2 Þ d2

In this formula, P1 is the expected incidence of the target infection in the comparison group; P2 is the expected incidence in the group receiving the experimental vaccine; d is P1
P2; Za is the Z score taken as the threshold for declaring the difference in incidence as ‘‘statistically significant’’ (e.g., 1.96 for a twotailed p value of 0.05); and Zb is the Z score for b error, the maximum tolerated probability of missing a significant difference when one really exists (e.g., 0.84 for a probability of 0.20). Because enrollment of a suitably large sample is a prerequisite for any trial, the variables and parameters specified by this formula provide a useful inventory of information that must be known or about which decisions must be made in planning a vaccine trial. To estimate P1, it is necessary to inspect earlier surveillance data for the target infection in the population contemplated for the trial. Predicting P1 can be particularly challenging for several reasons. First, for most infectious illnesses, a single year of surveillance will not be sufficient to estimate the expected incidence with confidence, as substantial year-toyear variation in incidence is common. For example, in Matlab, Bangladesh, where surveillance for cholera has been maintained for over 40 years, variations in annual incidence have been over 25-fold (50). Because it is ethically imperative that trials be designed to maximize the likelihood of yielding statistically meaningful results, it is always desirable to be conservative in projecting the likely incidence of the target infection in the comparison group (51). Second, because the expected incidence may differ dramatically, depending on demographic and other characteristics, it is important that estimates for the calculation be made for the group meeting the eligibility criteria for the trial. Third, investigators planning a trial must usually rely on earlier incidence data from routine public health surveillance, rather than from the special, prospective surveillance system that is usually instituted for a trial. Relatively loose clinical and microbiological criteria for defining infections are usually used in routine surveillance, tending

to elevate the observed incidence in relation to that detected with the much stricter diagnostic criteria employed in the prospective surveillance of a trial. Conversely, during a clinical trial, efforts are usually made to evaluate all patients who might have the target infection, many of whom would have been missed in routine surveillance, and to deploy diagnostic tests systematically on all such patients. This feature of surveillance would tend to elevate the observed incidence during phase III trials in relation to that observed with routine surveillance. Because of the unpredictability of the net balance between the factors that would inflate and factors that would diminish the incidence in a phase III trial, in relation to antecedent routine surveillance, conservatism is required in projecting incidence rates derived from earlier routine surveillance to be expected in a phase III trial. Yet a fourth consideration in estimating P1 is that volunteers enrolled for a trial usually are not representative of, and often have lower rates of infection than, the population from which they are drawn. For example, in a field trial of killed, oral cholera vaccines in Bangladesh, placebo recipients had a nearly 20% lower incidence of cholera than age- and gender-eligible persons who did not participate in the trial (52). Fifth, projections about P1 for a trial must account for anticipated subanalyses. Many trials are designed not merely to estimate vaccine protection in the entire study population but in subgroups of the population, such as those defined by age or other demographic variables. If such subgroups are of interest, the expected incidence of the target infection in each subgroup must be considered in designing a trial. Moreover, many pathogens have different phenotypes that may affect vaccine protection. For example, a field trial of killed, oral cholera vaccines found that the biotype of V. cholerae 01 (El Tor vs. classical) was an important modifier of vaccine protection (53). If different phenotypes of a target pathogen circulate in the population for a trial and if phenotype-specific vaccine protection is to be measured, P1 must be estimated separately for each of the different phenotypes of interest in designing the trial and estimating sample size requirements. Once the incidence for the comparison group has been estimated, P2 is readily calculable from the formula for PE. Therefore, investigators must determine what minimum level of PE should be detectable in the trial. Depending on the perspective of the trial, either of two strategies can be adopted. In a biologically oriented efficacy trial, evidence from earlier studies can be marshaled to arrive at a best guess about the likely level of protection. In a more pragmatically oriented effectiveness trial, the decision rests more on a minimum level of protection and a minimum duration of this protection that would be required to justify introducing a vaccine into public health practice. Such decisions must rely on quantitative analysis of numerous factors, such as the burden and costs of the target infection, the costs and side effects of the new vaccine, and the costs, benefits, and risks of alternative methods of prevention and treatment (54). The minimum level of PE to be detected must also take into account the planned strategy of analysis, as described earlier. If only persons who receive a complete course of appropriately administered vaccine are to be analyzed, as is often done in efficacy trials, estimates of protection can be based on the expected intrinsic potency of the vaccine. Conversely, if an intent-to-treat strategy is employed, whereby persons are analyzed according to their assigned agent regardless of whether the assigned agent was administered as

Chapter 5: Long-Term Evaluation of Vaccine Performance

intended, estimates of anticipated protection will have to account for the vagaries of incomplete and improper dosing that will inevitably occur in a proportion of participants. Decisions about Za address what p value will be considered as the threshold of statistical significance for rejecting the overall hypothesis of no PE. These decisions must also address whether statistical tests used to estimate p values will be interpreted in a one- or two-tailed fashion. A p value of 0.05 is the traditional, if somewhat arbitrary, threshold for declaring that a difference in incidence between compared groups is unlikely to have arisen by chance. However, as described in more detail in the section ‘‘Issues in Analyzing Data,’’ a more conservative p-value threshold may be desirable for a trial in which multiple analyses are planned (55,56). One-tailed tests may be appropriate when differences in the occurrence of the target infection are of interest only if they occur in one direction (e.g., with a higher incidence in one group vs. another group) and if a difference in the other direction would be of so little interest that it would not be statistically appraised. For a onetailed p value, Za will be substantially lower than that for the same probability interpreted in a two-tailed fashion, so this decision has important consequences for required sample sizes. A decision must also be made about Zb, or the Z score corresponding to the maximum tolerated probability of missing a significant difference if one really exists (57). Probabilities for b error are always interpreted in a one-tailed fashion; by convention, a b error 0.20 (Z  0.84) is considered acceptable. Although a complete discussion of sample size calculations is beyond the scope of this chapter, several additional points should be mentioned. The sample size must reflect the finally analyzed denominators. If these consist only of persons who receive a complete regimen of an assigned agent, it will also be important to consider the source population from which this final sample is assembled. From persons who meet the age and gender restrictions for the trial, some will be considered ineligible because of other exclusionary criteria, some will refuse, some will be absent at the time of vaccination, and some will receive the assigned agents in an incomplete or erroneous fashion. In addition, after initial assembly of participants, subjects may be lost to follow-up or may otherwise have their follow-up terminated, as described earlier. These events yield a population effectively under follow-up that is lower than the source population, necessitating careful and conservative planning in the selection of the source population (48). For example, in the field trial of killed oral cholera vaccines in Bangladesh, only 32% of the source population ultimately received a complete course of vaccine or placebo and were followed for the first year after dosing (51). If vaccine efficacy is to be estimated for subgroups, it will be necessary to estimate the proportion of participants who are likely to be included in each subgroup and to calculate an overall sample size large enough so that a statistically meaningful comparison will be possible for each subgroup of interest. If differences in vaccine efficacy between subgroups are to be evaluated, sample sizes substantially larger than those required for assessments of subgroup-specific efficacy will usually be required (58). Finally, sometimes merely rejecting the null hypothesis of no vaccine efficacy does not provide an adequate assurance that a vaccine is good enough. For example, in response to the troublesome side effects of conventional whole-cell pertussis vaccine, acellular pertussis vaccines have been developed and tested. To be considered suitable for licensure, these vaccines

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must, in addition to being safe, be as protective as conventional vaccines. Sample size considerations of trials comparing acellular vaccines with conventional vaccines, therefore, had to demonstrate that the PE of acellular vaccines was not substantially inferior to that of conventional pertussis vaccines (59). Demonstration of non-inferiority of one agent in comparison with another requires a different approach to the calculation of sample size requirements. Instead of seeking to reject the null hypothesis of no difference between the agents with adequate power, this approach seeks to assure that the confidence interval for the difference in outcomes between the agents excludes values suggesting that the new agent (e.g., acellular pertussis vaccine) is unacceptably inferior to the standard agent (e.g., conventional pertussis vaccine).

Issues in Allocation of the Compared Agents Randomized allocation is such an important safeguard against bias that it should be regarded as essential in field evaluations of new vaccines. However, even if randomized allocation is planned, several issues still require attention. An obvious decision to be made is whether the allocation procedure is to yield groups with similar or intentionally unequal numbers of subjects. Whereas groups of similar size are usually preferred in two-group trials, the decision is less straightforward for trials with more than two groups, particularly when a single nonvaccinated group is to be compared with multiple vaccinated groups. For such multiple-group trials, there is disagreement about whether equal or unequal sample sizes provide optimal statistical efficiency (60). Moreover, on ethical grounds, there may be compelling reasons to minimize the number of participants in the group that will not be vaccinated. If unequal sizes per group are desired, randomization procedures can easily be adapted to yield the desired allocation ratio. Stratified allocation, wherein subjects are randomly allocated to different agents within subgroups defined by relevant risk factors for the target infection, typically in a balanced fashion (with use of ‘‘blocking’’) within these strata, has been advocated as a method to improve the similarity of baseline characteristics of compared groups (61). This technique is helpful in safeguarding against bias in trials involving small numbers of participants, particularly when these participants differ greatly in their risks for the outcome under study. Such conditions commonly apply to clinical trials of therapy for ill persons who have substantially different prognostic expectations. This technique may also be relevant to vaccine trials with small sample sizes, but it is unlikely to confer great advantage over the use of simple randomization in assigning compared agents for large-scale field trials (62). Blocked allocation is also commonly employed in small trials in which there is concern that simple randomization may not yield groups of the desired size. For example, in a twocelled trial of vaccine versus placebo intended to compare groups of equal sizes, randomization might take place within blocks of every four consecutively assigned subjects to ensure that two subjects receive vaccine and two receive placebo (62). As described earlier, it is conventionally recommended that randomization of subjects should occur only after enrollment procedures for participation have been completed. However, in practice, this prescribed sequence may be a difficult requirement to fulfill, particularly in less developed settings,

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where communications may be inadequate to permit a vaccination team to contact a central randomization unit and where it may not be desirable to involve field teams in the task of allocation (23). In such circumstances, it may be preferable to allocate the agents in a randomized fashion before recruitment and ascertainment of eligibility and to safeguard against biases in the enrollment of participants by administration of the compared agents in a double-blinded fashion. With this strategy, each member of an already characterized population can be randomly preassigned an agent, as in a field trial of oral cholera vaccines in Bangladesh (45), or doses of agents can be randomly ordered, either physically within storage containers or by means of a coded assignment list, and the doses can be given consecutively as participants are enrolled into the study. Another issue requiring consideration is the unit of participants to be allocated to the compared agents. From a statistical viewpoint, it is most efficient to consider the individual participant as the unit to be allocated (48). However, allocation of individuals may be unwise for vaccines that are excreted and transmitted from a vaccinated individual to nonvaccinated contacts, and, as noted earlier, it may not be applicable when an effectiveness rather than an efficacy perspective is selected. To evaluate the protection conferred by a transmissible vaccine, it is necessary to demarcate as units for allocation individuals between whom transmission of the vaccine is unlikely. For example, in a trial of orally administered, live Ty21 a typhoid vaccine in Chile, classrooms of students were allocated to the compared agents, in part to safeguard against the possibility of ‘‘contamination’’ of nonvaccinated controls by transmission of the vaccine organism (38,40). When calculating sample sizes in trials in which a group is the unit of allocation, it is important to account for the degree of clustering of the target infection within the groups to be allocated. Cholera, for example, occurs in highly focal outbreaks within a community, even in endemic area (50). In general, the greater the magnitude of such clustering, the larger will be the number of subjects who will be required for the evaluation. In addition, when groups rather than individuals serve as units of allocation, statistical techniques appropriate for this allocation strategy will be required in analyzing the results of the trial (63). Finally, in addition to providing safeguards against biased allocation, it is necessary to measure whether the allocation created groups that were comparable in baseline characteristics that might affect the occurrence of the target infection (19–21). Documentation of equivalent distributions in the compared groups reassures that the randomization procedure was appropriately executed, although some imbalances in the distributions of baseline features may be expected by chance (22). Adjustment of estimates of vaccine PE for unequally distributed variables can be undertaken with stratified analyses or multivariate models. Comparison of these adjusted estimates with the crude estimates provides an indication as to whether distortions of vaccine protection could have arisen because of unequal baseline susceptibility to infection in the compared groups (22). In addition to baseline characterization of participants, it is important to document reasons for nonparticipation and baseline characteristics of the nonparticipants. Such data permit assessment of whether participants were representative of the target population; if subjects were preassigned to different agents, these data can be analyzed to evaluate whether differential participation of those assigned to the compared groups was a likely source of bias.

Issues in the Administration of the Compared Agents Several questions routinely arise about how the agents should be packaged and labeled as well as about what data should be collected concerning the process of administration. Agents can be packaged as single doses or as containers with multiple doses. In general, it will be advantageous to package the agents in single doses, both to minimize vaccine deterioration in the field and also to circumvent human errors in measuring doses. If the agents are to be coded, as is necessary to maintain double blinding, several factors must be considered: simplicity, avoidance of errors in administration, and prevention of discovery of the identities of the agents. Whereas a unique code for each dose, usually with a number, provides the greatest protection against the unblinding of a trial, this strategy may increase the complexity of administering agents and of recording what was administered, and it may also create logistical difficulties if the same agent is to be given in a multidose regimen. Use of fewer codes may alleviate these problems but may make it easier for participants or investigators to detect the identities of the codes. Whatever system of coding is employed, letter or number codes are substantially simpler to work with than color codes. Because of the inevitable tendency of research workers to be inquisitive about the identities of the coded agents and because of the difficulties in making truly identical agents for comparison in vaccine trials, it is also useful at the end of a trial to conduct a survey of investigators and other workers in the trial to assess opinions about the identities of the different codes. This exercise, which will permit an empirical estimate of the likelihood that the trial was indeed conducted in a doubleblinded fashion, is particularly valuable if only a few different codes are used for the compared agents. Several additional aspects of the administration of the agents require documentation. First, even if the agents are preassigned, it is important that vaccination teams record the code of what was actually given. If single-unit doses are administered, it is useful to employ self-adhesive stickers that give the code and that can be removed from each dose and affixed to a vaccination record book. This documentation is important, because errors in administration are inevitable. For example, in a trial of oral cholera vaccines in Bangladesh (45), 573 of 234,032 (0.2%) doses were not given as assigned, despite use of a simple A-B-C coding system for the three compared agents. Second, it is desirable to record observations about the completeness of dosing. Incomplete dosing can occur with any route of administration, but it may be a particular problem for noninjectable agents. In the Bangladesh oral cholera vaccine trial, 6367 (3%) doses were not completely ingested, in part because of the large volumes (50–165 mL, depending on age) of the doses (45). Although analytical strategies for handling participants who received erroneous or incomplete doses may vary according to the perspective of the trial, documentation of the frequency of such vagaries is always helpful in interpreting the apparent degree of vaccine protection. If the agents under investigation require a cold chain to preserve potency, it will be desirable at each step in the itinerary, from the manufacturer to the recipient, to document that necessary thermal conditions were maintained. A variety of thermal monitors are available for this purpose. If a particular level of an immune response to vaccination is known to occur after proper administration of fully potent vaccine, assessment of immune responses in representative samples of the vaccinated and comparison groups may provide a useful

Chapter 5: Long-Term Evaluation of Vaccine Performance

indication of the adequacy of the manufacture, storage, and administration of the vaccine. Finally, samples of the batches of vaccine delivered to the field site for use in the trial should be preserved for evaluation of potency. All of these tactics are of particular interest for trials in which protection by vaccination is lower than expected, and it is necessary to distinguish deficiencies in the intrinsic potency of a vaccine from problems in manufacture, storage, and administration.

Issues in Surveillance The scope of surveillance activities in a trial may include detection of the target infection and other clinical events relevant to the assessment of vaccine protection, observations of adverse effects, assessment of immune responses, and recording of competing events. For each of these outcomes, decisions must be made about the approach to detecting events, the time frame for surveillance, as well as the methods to be employed to safeguard against biased detection of the events. Scope The range of detected events for assessment of vaccine protection may vary substantially with the method of detection. Passive surveillance of illnesses through monitoring of routine visits by patients to health facilities provides a logistically simple approach to detecting outcomes. However, with passive surveillance, only illnesses that are severe enough to prompt solicitation of medical care will be detected and persons who have more ready access to treatment facilities or who are more ‘‘medicalized’’ in their use of health care facilities may be overrepresented. Moreover, the type of events detected may be affected by the focus of the treatment facility under surveillance. For example, a trial that uses infectious diseases hospitals to detect outcome events may miss neurological or cardiovascular adverse effects following vaccination. With an active surveillance strategy, the investigator maintains a schedule of contacts with each participant, regardless of whether the participant is ill or not, and thereby has the opportunity to detect illnesses that span a wide spectrum of severity and characteristics. In addition, with active surveillance, it may be possible to detect asymptomatic infections, whose interruption may be relevant to preventing transmission of disease. The salient disadvantage of active surveillance, however, is the considerable expense and logistical complexity of maintaining regular, active contact with an entire study population, particularly in studies that are large or that entail a prolonged period of follow-up. For certain types of target infections, active surveillance will yield a greater apparent incidence than that noted by passive surveillance, thereby reducing sample size requirements for evaluation of vaccine protection. For example, in Matlab, Bangladesh, which has been a site for several cholera vaccine field trials, comparison of active and passive surveillance data for children aged under two years demonstrated that only about 6% of diarrheal illnesses detected by active surveillance were brought to treatment facilities for care (64). However, investigators specifically interested in the effect of vaccination on clinically severe disease should remember that the overall incidence rates based on active surveillance may reflect primarily nonsevere infections. This difference in clinical spectrum may greatly affect the magnitude of vaccine PE if a vaccine acts to diminish the severity of the clinical manifestations of an infection, as has been noted for whole-cell pertussis vaccines (65) and inactivated oral vaccines against cholera (66).

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For such vaccines, estimates of vaccine efficacy based on passive surveillance are likely to be higher than estimates based on active surveillance. Thus, choices between active and passive surveillance in a trial should not be based merely on trade-offs between logistical ease and disease incidence but should consider the possible impact of the surveillance technique on the magnitude of vaccine protection. Depending on the research question posed by the trial, passive surveillance, active surveillance, or a combination of the two may be appropriate for detection of target infections. Detection of adverse events following dosing, and comparing rates of specific adverse events in the groups under study are essential components of a phase III trial. Indeed, phase III trials, because of their large size, offer a unique opportunity for evaluating vaccine safety before a vaccine is licensed. In the past years, the approach taken by phase III trials was often to focus on documentation of frequent and expected common adverse effects, occurring only during the first few days after dosing. The focus was primarily on documenting common side effects, generally of mild severity. Often these trials measured adverse events in only a small subsample of the trial population, as large sample sizes were not required to document frequent side effects, such as the occurrence of pain or erythema at an injection site. Some trials failed to conduct surveillance for adverse events altogether. Several changes have occurred during the past several years in the approach to documenting vaccine safety in phase III trials. First, it is now well recognized that not all adverse effects of vaccination are easily predicted. For example, intussusception following oral receipt of live rhesus rotavirus reassortant vaccine, an event that is now well documented, was not an expected side effect of this vaccine, at least at the time when phase III trials were undertaken (67). It is therefore inappropriate in phase III trials to constrain the focus of surveillance to adverse events that can readily be predicted. Second, it is no longer acceptable to target surveillance only to adverse events that occur quite commonly nor to place only a subsample of participants under surveillance for adverse events. Two recently licensed live oral rotavirus vaccines, for example, were tested in phase III clinical development programs, each of which enrolled over 50,000 infants, to exclude a very modest attributable incidence of intussusception in vaccine recipients (68,69). Expanding the scope of surveillance in this fashion will have a major effect in increasing the size, cost, and complexity of future phase III trials. Surveillance for immune responses will almost always require actively scheduled tests, although immune testing of vaccinated participants who develop target infections and are detected in ordinary treatment settings may offer the opportunity to evaluate whether poor immune responses accounted for the illness in these vaccines (70). If the goal of these immune assessments is merely to confirm that the vaccines under study elicited expected levels of immune responses, it may be necessary only to evaluate a sample of the trial population. However, phase III trials also commonly attempt to evaluate the relationship between the magnitude of induced responses and the level of vaccine-induced protection. To achieve the latter objective, responses in vaccinees who ultimately developed the target infection are compared with responses of vaccinees who did not. Since breakthrough infections are usually small in number and since individuals who develop these infections cannot be predicted at the time of dosing in a trial of a protective vaccine, it will often be necessary to obtain post-dosing specimens for

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immunological evaluation from most or all subjects in a trial to enable a statistically satisfactory evaluation of immunological correlates of protection. Detection of deaths and out-migrations is important to enable calculation of the person-time at risk during follow-up of trial participants. Detection of these events may be possible by consulting vital event data routinely collected for the population under study, but detection of losses for other reasons (e.g., refusal to continue participation) as well as adequate characterization of the reasons for losses to follow-up will usually require an active strategy. Because the assessment of cause-specific mortality should constitute an element of surveillance for adverse events in any phase III trial, arrangements must be made to obtain clinical records of illnesses leading up to all deaths in trial participants or, lacking such data, it is important that relatives of decedents be interviewed to obtain ‘‘verbal autopsies’’ (71). Time Frame The time frame for surveillance must be planned to address the questions posed by the trial. Because field trials provide the best opportunity to evaluate the duration of protection conferred by vaccination, it will usually be desirable to plan for the possibility of long-term surveillance. Investigators should not, however, be lulled into the intellectual trap of extending the period of surveillance merely as a mechanism to raise the cumulative incidence of the target infection and thereby to reduce sample size requirements. If short-term efficacy is of interest, sample size requirements should be calculated on the basis of the expected incidence for the shortest duration of follow-up to be analyzed (51). It is now appreciated that vaccine side effects can occur long after vaccine dosing. For example, quite unexpectedly, mortality rates during the third year of life were found to be elevated in female infants in less developed countries who received high-titered measles vaccines at six months of age (72). As a result, national regulatory authorities are increasingly demanding that phase III trials be designed to capture adverse events of any grade of severity: those that are predictable as well as those that are not; those that occur at any point during the follow-up of subjects in the trial, not just the first several days after dosing; and those that occur infrequently. Similarly, although short-term immune responses are often measured to gauge whether the vaccine induced expected levels of immune responses and to assess immune correlates of vaccine protection, serial surveys of immune responses may estimate the kinetics of decay of immunity over time. The duration of a trial may be truncated if severe side effects are noted during vaccination, contraindicating further dosing, or if a large health benefit occurs in a group that receives an active agent, so that ethical considerations demand that participants randomized to other agents have the opportunity to receive the beneficial agent. The latter consideration is likely to be a major factor in the design of future trials of vaccines against human immunodeficiency virus, particularly if a vaccine that reduces the rate of infection is identified (73). Since investigators may have a vested interest in prolonging a trial to obtain estimates of long-term protection, it is desirable to place such decisions in the hands of monitoring committees of scientific peers who are not in any way involved in the trial and who can make decisions purely on behalf of the subjects in the trial (74). Modern phase III trials are usually monitored by

at least two bodies external to the investigative team: a data and safety monitoring board (DSMB) and an institutional review board (IRB). The DSMB is typically composed of professionals with expertise in disciplines relevant to the trial, such as biostatistics, epidemiology, and clinical infectious diseases. These individuals cannot have any role as investigators in the trial. Typically, a DSMB has the authority to review and approve the final protocol for the trial. It also serves to monitor the trial periodically by assessing both the performance of the trial in meeting its process goals, such as subject enrollment, and by evaluating the adverse events and study end points that are observed among trial participants. In these activities, the board is responsible for making recommendations, including early termination of the trial, if need be, to the sponsor of the trial. Detection Bias Detection bias distorts estimates of vaccine efficacy via unequal surveillance of vaccinees versus controls for the target outcome. This bias can occur in at least three ways. First, if there are unequal losses to follow-up of study participants in the compared groups, there may be an unequal opportunity to detect outcome events (23). Although analytical techniques, such as life table analyses, are designed to adjust estimates of protection for unequal periods of follow-up in compared groups, such techniques may not adequately correct for a bias that may occur if the reasons for the losses and the characteristics of the lost subjects differ in the compared groups (16,17). Losses to followup are less likely to create a bias if they are few in number and if decisions to drop out of the study are not based on knowledge of which agent has been received. Therefore, protection against this bias is best accomplished by choosing a study population that is likely to comply with the study protocol and to have a low rate of migration, by vigilant efforts to maintain contact with the study population and by double blinding of the trial. Moreover, it is essential that the numbers and characteristics of subjects who are lost to follow-up, as well as the reasons for the losses, be analyzed for each group to permit judgments about whether or not unequal losses in the compared groups were likely to have created a bias. Second, if subjects in the compared groups have an unequal probability of receiving diagnostic procedures that are necessary to detect outcome events, the comparative occurrence of the events in the groups may be distorted (23). Doubleblinded surveillance provides the most effective safeguard against this possibility. However, effective use of this safeguard will not always be possible in vaccine field trials. For example, in trials of BCG vaccination against tuberculosis and leprosy, it was not ethically permissible to employ a comparative agent whose side effects, including creation of a cutaneous scar, were similar to those of BCG (75,76). For trials that are not conducted in a double-blinded fashion, it is important that investigators employ alternative safeguards against biased diagnostic evaluations or at least that they evaluate whether a differential intensity of diagnostic testing of subjects in the compared groups was likely to have distorted estimates of vaccine protection (23). For example, in field trials of BCG against tuberculosis, use of mass radiographic screening for pulmonary disease, irrespective of the presence of symptoms and of solicitation of clinical care, provided an important safeguard against biased application of diagnostic tests (75). Third, even if bias has not resulted from differential losses to follow-up or from differential diagnostic surveillance,

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biased detection of outcomes can still occur if the evidence collected during diagnostic evaluations has not been interpreted in an objective fashion. In a truly double-blinded trial, bias caused by differential diagnostic ascertainment is not a major risk. In a trial in which double-blinded surveillance is not possible, alternative arrangements must be made to ensure objective diagnoses. For example, to prevent biased diagnoses in trials of BCG vaccination against leprosy, a useful tactic was to cover the injection sites of both vaccines and nonvaccinees during physical examinations (76). Similarly, in trials of BCG against tuberculosis in which mass chest radiography was used to detect potential cases of tuberculosis, an additional safeguard against biased diagnoses was provided by blinding radiologists to the vaccination status of subjects whose chest films were under evaluation (23). In addition to such tactics for preventing biased diagnoses, it is also necessary to maximize the accuracy of diagnoses by ensuring that the diagnostic data have been collected in a systematic and accurate fashion and that appropriate and explicit diagnostic criteria have been uniformly applied to the collected data. Diagnostic inaccuracies, even if they occur in a random fashion, may substantially distort measures of the comparative occurrence of events in the groups under study (77). Finally, in evaluating the possible role of detection bias in distorting estimates of vaccine PE, it may be helpful to inspect vaccine protection against an indicator condition—a disease whose diagnostic evaluation is similar to that for the target infection but against which no vaccine efficacy can logically be anticipated. If the vaccine under evaluation fails to protect against such a condition, the likelihood of detection bias is diminished. For example, in the trial of inactivated oral cholera vaccines in Bangladesh, it was demonstrated that neither vaccine conferred protection against bloody diarrhea, as had been predicted by expectations that protection would occur primarily against diarrhea due to V. cholerae 01 and enterotoxigenic Escherichia coli but not against invasive enteropathogens (78).

Issues in Analyzing Data Although a detailed discussion of statistical strategies for analyzing data in vaccine field trials is beyond the scope of this chapter, a few general comments deserve emphasis. It is important that groups under analysis be assessed for their comparability at baseline (e.g., at allocation), and that the comparability of losses to follow-up be evaluated after baseline. If imbalances are detected, appropriate analytical techniques should be employed to check if the results might have been distorted by baseline and post-baseline imbalances and to correct denominators for losses to follow-up (22). It is also important that analyses be planned in advance of the study. Before inspection of the data, explicit criteria should be developed for determining which participants will constitute the groups under analysis, and detailed criteria should be formulated for defining outcome events in these analyzed participants. Because the design and interpretation of analyses may be distorted by biases of the data analyst, it has also been recommended that analyses be undertaken and interpreted without knowledge of the agents received by the compared groups (18). It is unwise for investigators to continuously inspect the occurrence of outcome events in the different groups as the data accumulate. The dangers are several. First, if the comparison group for the study receives an agent that is known to be

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inactive against the target infection and if vaccinated groups are protected, the observed differences in rates of the target infection may ‘‘unblind’’ the investigation and jeopardize the scientific quality of data as surveillance continues. Second, the apparent degree of protection by a vaccine may fluctuate widely with time. For example, during the initial years of follow-up in a trial of oral Ty21a vaccine against typhoid in Area Norte of Santiago, Chile, efficacy fluctuated from 0% to 100% during various three-month intervals, although there was no consistent trend over time (39). If investigators use accumulating surveillance data to determine intervals for evaluating ‘‘short-term’’ and ‘‘long-term’’ protection, such fluctuations may lead to the choice of intervals that severely distort estimates of protection. These considerations do not, of course, argue against ongoing inspection of data by an independent monitoring committee, for the ethical reasons outlined earlier, nor against formally planned interim analyses of the data. However, because of the danger of spurious conclusions that can be arrived at during multiple looks at the data (74), it is important that suitably conservative statistical strategies be used to evaluate the data. Multiple analyses of accumulating data constitute one facet of the more general multiple-comparisons problem, alluded to earlier (22,55,56,74,79). The multiple-comparisons problem arises when investigators undertake intergroup comparisons of several outcomes or if the intergroup occurrence of an outcome is compared between several pairs of agents (if more than two are under study), within several subgroups of participants, or at multiple points in time during follow-up. As more analyses are conducted, the overall probability of finding at least one ‘‘statistically significant’’ difference when no true difference exists also increases. For example, if p < 0.05 is the threshold for declaring a difference significant, this means that the investigator is willing to tolerate a 1:20 chance of finding a statistically significant difference arising from chance fluctuations when in fact there is no difference between the groups under analysis. If 20 independent comparisons are evaluated, the overall probability that at least one will be statistically significant at p < 0.05, even if no difference exists, will be 0.64, not 0.05. To compensate for this problem, several statistical techniques have been developed for reducing the p value chosen as the threshold for declaring an individual comparison as statistically significant (55,79). However, because such techniques create progressively smaller p-value thresholds as the total number of comparisons increases, the application of these methods in trials with numerous analyses would make it difficult or impossible to detect statistically significant differences. Use of these techniques is proper only for analyses that are anticipated in advance rather than being suggested after inspection of data (55,56). These and other dilemmas have created controversy about the proper approach for dealing with multiple comparisons in the analysis of RCTs (56). Although many strategies are possible, one approach would be to pose primary analyses, addressing the major questions of the trial, and secondary analyses, evaluating other topics of interest. Adjustments of p values for multiple comparisons would be made only for the primary analyses, and the results of these analyses would be considered as rigorous hypothesistesting assessments. Secondary analyses would employ ordinary (e.g., p < 0.05) p-value thresholds but would be conducted as hypothesis-generating exercises requiring conservative interpretation and future replication in additional studies (45).

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Finally, an increasing emphasis is being placed on the use of confidence intervals in statistical evaluations of vaccine efficacy, rather than merely declaring PE as ‘‘statistically significant’’ at some arbitrary threshold (e.g., p < 0.05) (80). This is because a statistically significant result for vaccine protection merely implies that the results for PE reject the null hypothesis of no vaccine protection; declaration of statistically significant PE provides no information about the range of values for protection that are statistically compatible with the observed level of efficacy. For the latter, the confidence interval surrounding the estimate of efficacy at a desired level of precision (e.g., 95%) must be calculated. In trials aimed at detecting whether or not the vaccine is protective, the lower boundary of the confidence interval portrays not only whether the results reject the null hypothesis (e.g., do not include 0% efficacy) but also how low PE might really be, allowing for the play of chance in the trial’s results. For trials designed to determine whether the efficacy of a tested vaccine is at or above some predetermined level, the lower boundary tells whether or not the tested vaccine’s true efficacy is likely to be at or above the desired level (59).

Good Clinical Practice In recent years, regulatory agencies have placed much emphasis on the concept of conducting vaccine trials leading to vaccine licensure with designs and procedures that conform to ‘‘Good Clinical Practice’’ (GCP) (81). The elements of GCP are designed as basic criteria to ensure that trials are conducted in an ethically justifiable fashion, are scientifically sound, and yield data that are verifiable. The last feature refers to the need to create sufficient documentation during a trial so that an independent auditor could verify that the findings of a trial accurately reflect the data actually collected.

POST-LICENSURE OBSERVATIONAL STUDIES After a vaccine has been licensed, it is still necessary to monitor its safety and the protection it confers in practice. Various types of studies are commonly used to evaluate new vaccines once they have been introduced into practice. These include serological assessments of vaccine-induced immunity, evaluations of the levels of vaccine coverage of the intended target population, and assessments of vaccine safety and protection. A detailed consideration of all of these studies is beyond the scope of this chapter. Here we consider why it is necessary to conduct postlicensure studies of vaccine safety and clinical protection and briefly consider several of the more common designs for clinical studies of these issues.

Reasons for Conducting Post-licensure Studies of Safety and Protection The reasons for continued vigilance in monitoring clinical safety and protection after licensure of a vaccine are several (82). The spectrum of vaccine recipients in practice may expand beyond that studied in phase III trials, and this expansion may lead to a decline in vaccine protection. This phenomenon has, for example, been noted for conventional influenza vaccines, which often perform better in the young, healthy subjects often used for testing than in elderly patients, who constitute one of the primary targets for these vaccines (83). Alternatively, the target population may not change, but administration of the vaccine may later be found to be less than optimally focused. For example, long after conventional measles vaccine was put

into practice in the United States, it was discovered that administration of this vaccine at 12 months of age accounted for some vaccine failures, leading to a later recommendation that the vaccine be given at 15 months (84). The vagaries of manufacturing practice may lead to the inadvertent release of lots of vaccines that are not fully protective or are harmful. The former was illustrated by problems with the immunogenicity of certain post-licensure lots of PRPOMP, a Hib capsular polysaccharide—Neisseria meningitidis outer membrane protein conjugate vaccine (85), while the latter was perhaps most dramatically illustrated by the Cutter incident, in which inadequately inactivated lots of the Salk polio vaccine caused paralytic polio in U.S. vaccine recipients in the 1950s (86). Even without manufacturing errors, the formulation or dosing regimen of a vaccine may ultimately prove unsuitable when the vaccine is administered on a large scale. Sabin oral polio vaccine is less immunogenic when given to infants residing in less developed settings. This observation led to the conclusion that more doses of the polyvalent vaccine, or even the use of monovalent vaccine, may be required to protect infants and children in these settings (87). Similarly, the titers of the three serotypes contained in the Sabin vaccine had to be modified when post-licensure studies found that the vaccine failed to confer suitable protection against type 3 infections in certain developing countries (88). Appropriately manufactured vaccines may be responsible for rare or long-latency adverse effects that are not detectable with pre-licensure phase III trials and appear only when the vaccine has been administered to larger numbers of persons over long intervals of time. Although pre-licensure studies showed an inactivated vaccine against swine influenza to be safe, a putative association between vaccination and the rare development of Guillain-Barre´ syndrome emerged when the vaccine was applied on a mass scale (89). The vagaries of public health practice may lead to errors in vaccine storage or administration that may vitiate vaccine protection. This phenomenon was noted for earlier generations of conventional measles vaccine, for which storage at unacceptably high temperatures led to reductions of vaccine potency (90). Agents coadministered with vaccine in practice may cause unexpected reductions of vaccine potency. This problem was noted for human diploid-cell rabies vaccine, whose immunogenicity is reduced by concomitantly administered chloroquine, explaining an unexpected vaccine failure in a Peace Corps volunteer who took both agents (91). Finally, the mass administration of a vaccine may lead to unexpectedly higher levels of vaccine protection than were expected on the basis of phase III trials, owing to the herd protective effects of vaccination in reducing transmission of the target pathogen. This phenomenon was noted for PRP conjugate vaccines against invasive Hib disease; despite moderate levels of coverage of targeted children in several industrialized countries, mass administration of these vaccines has virtually eliminated invasive Hib disease in these settings (92). Although this cannot be classified as a post-licensure ‘‘problem,’’ it is important to document these indirect vaccine effects.

Methodological Approaches After licensure, it is sometimes appropriate to evaluate vaccines with RCTs. For example, it may be of interest to compare the performance of different licensed vaccines with one another or

Chapter 5: Long-Term Evaluation of Vaccine Performance

different regimens of such vaccines. Such an evaluation was reported in the ‘‘mix and match’’ study of different licensed PRP conjugate vaccines, in which vaccine safety and immunogenicity were assessed in subjects randomized to various multidose regimens of the same vaccine or to different vaccines interchanged with one another within a given regimen (93). Often, however, it will not be ethically permissible, logistically and financially feasible, or scientifically appropriate to use RCTs to evaluate a vaccine after it has been licensed (13,14). Ethical problems arise in any design in which an indicated vaccine is withheld from study subjects who are experimentally allocated to an inert control agent. In addition, because of the expense of RCTs, it is not possible to consider performing a new trial for each question that arises about a vaccine after its introduction to practice. Scientifically, the questions posed after licensure usually address the performance of the vaccine as it is actually given in practice, and it may be difficult to fully replicate practice with a trial, even using the effectiveness trial approach outlined in the section ‘‘Clarification of the Perspective of the Trial.’’ For these reasons, observational study designs are most commonly used to evaluate the safety and protection of a vaccine used in practice. These studies are called observational rather than experimental because the investigator does not allocate subjects to receive alternative agents according to some deliberate plan, such as randomization, but assesses the outcomes of receipt or nonreceipt of vaccine as occur during routine practice (94). Since clinical outcomes under study can be either adverse events or the target illnesses to be prevented by vaccination, these allow assessment of both vaccine safety and vaccine protection. Indirect Approaches: Before-and-After Studies Perhaps the simplest way to evaluate a vaccine applied in practice to a population is simply to monitor the incidence of the outcome—a side effect or the disease to which the vaccine is directed—before and after the vaccine is introduced. Such evaluations have, for example, provided convincing evidence about the effectiveness of PRP-protein conjugate and measlesmumps-rubella vaccines as well as oral polio vaccines, for which mass immunization has nearly or completely eliminated the target diseases (92,95,96). When the overall incidence of the disease outcome before and after initiation of a vaccine program is assessed, the study is an indirect evaluation of vaccine performance, since vaccinees are not directly compared with nonvaccinees to evaluate the vaccine. Instead, the evaluation of the vaccine relies on the temporal trend of disease in the entire target population regardless of the proportion of its members who became vaccinated. Apart from situations in which there are dramatic vaccine effects on diseases that, in lieu of vaccination, occur predictably year after year and in which disease surveillance is reliable and constant over time, such studies are difficult to interpret. This is because, without comparison of the experience of vaccinees with concurrently followed nonvaccinees, it is usually difficult to know whether temporal changes in the occurrence of disease outcomes reflect the effect of the vaccine, changes in disease epidemiology unrelated to the vaccine, changes in intensity or accuracy of surveillance for the disease, changes in other interventions that may modify the occurrence of the disease, or changes in diagnostic definitions or reporting of disease (94).

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Direct Approaches

Controlled, cohort studies. If we consider the alternative approach of directly comparing concurrently assembled vaccinees and nonvaccinees for disease outcomes, the most straightforward design is a controlled cohort study (6,82,94,97). In such a study, the compared groups are then assessed for the incidence of the studied outcome, determined through longitudinal surveillance after assembly. These compared cohorts can be assembled historically, in present time, or a mixture of the two. Similarly, the follow-up of the compared cohorts can occur during an interval prior to the investigation, pari passu with the investigation, or both. A major advantage of controlled cohort evaluations is that they permit comparisons of the cohorts for multiple disease outcomes within a single study. As a result, if properly designed, a single controlled cohort study can evaluate multiple potential side effects and multiple potential protective effects of vaccination. Another advantage over simple before-and-after comparisons is that cohort studies enable direct estimation of vaccine protection by the same expression, PE ¼ (1
RR)  100%, cited earlier for RCTs. This is because RCTs are themselves controlled cohort studies. However, controlled cohort studies have several limitations. For adequate statistical power to detect vaccine effects, such studies must detect a suitable number of disease outcome events. These studies are thus best reserved for situations in which the studied outcomes occur frequently, thus ensuring adequate statistical power to detect intergroup differences. These investigations are also better suited for evaluations of outcomes that occur relatively shortly after vaccination, so as to minimize the logistical complexities and financial expense of prolonged follow-up of a study population. Examples of research questions well suited to cohort studies include evaluations of vaccine protection against common childhood diseases, such as measles (98), and assessments of vaccine protection during defined outbreaks of the target disease, in which the study population experiences a high attack rate of disease. Because most post-licensure assessments of putative vaccine side effects focus on long-latency or rare events, cohort designs are not commonly used for such evaluations. Case-control studies. When the disease outcome under study is rare or occurs long after vaccination, it is usually more feasible to evaluate the vaccine using the case-control study design (6,82,99). With this design, groups are assembled not on the basis of being vaccinated or not, as in a cohort study, but on the basis of having developed the disease outcome (‘‘cases’’) or not (‘‘controls’’). Cases and controls are then contrasted for earlier receipt of vaccination. In a case-control study, cases and controls can be assembled with use of historical records, prospective surveillance, or a mixture of the two. However, histories of vaccination in these studies usually rely on historical information. Case-control assessments have several advantages. Because cases are sampled directly with these designs, casecontrol studies are well suited for studying rare disease outcomes of vaccination as well as outcomes that may occur with long-latency periods after vaccination. Moreover, because the investigator can arbitrarily select a statistically optimal number of controls for each case and can often enroll the required sample of cases and controls over a relatively brief period of time, the case-control design maximizes statistical power to detect outcomes associated with vaccination, minimizes sample size requirements, and substantially reduces the logistical complexity and financial expense of the investigation.

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The case-control design does not provide an estimate of disease incidence in vaccinees and nonvaccinees, therefore, the RR component of the PE formula is not directly calculable. However, when the disease outcome for the study is rare or when suitable sampling strategies are used for selecting cases and controls (100), the RR from a controlled cohort design is approximated by the odds ratio (OR) of vaccination in cases versus controls, where OR ¼ (odds of vaccination in cases)/ (odds of vaccination in controls) and PE ¼ (1
OR)  100%. Because cases are defined only on the basis of one disease outcome, a case-control study can evaluate only one outcome of vaccination. However, with a well-focused research hypothesis, case-control studies can enable powerful assessments of vaccine performance in practice. For example, recent case-control studies have provided useful post-licensure evaluations of vaccine protection against such rare diseases as invasive pneumococcal (5) and Hib (101) infections. In addition, this design has proved extremely useful in evaluating rare but serious potential side effects, such as Guillain-Barre´ syndrome following vaccination with swine influenza vaccine and serious pediatric neurological syndromes following vaccination with conventional whole-cell pertussis vaccine (89,102). Variant designs. It has been proposed that vaccine performance can sometimes be assessed in the context of prevalence surveys designed primarily to assess vaccine coverage of a target population (82). At the time of the survey, respondents are asked about the date of past vaccination as well as the date of intervening disease and vaccine protection is calculated as if a conventional controlled cohort study had been done on vaccinees versus nonvaccinees. This design differs from a conventional controlled cohort study in that it evaluates only those members of the original cohorts of vaccines and nonvaccinees who are still present at the time of the survey. It thereby ignores cohort members who have migrated out or died during the interval before the survey. Consequently, this design is sometimes referred to as a ‘‘residue cohort.’’ Routine statistics on vaccine coverage can also be used to serve as control groups in assessments of vaccine protection. It has, for example, been proposed that vaccine coverage ascertained for a representative series of cases can be compared with routinely available estimates of vaccine coverage for the source population for the cases as a tactic to monitor vaccine protection on a routine basis (103). ORs of vaccination in the case group relative to the source group are calculated, and the expression (1
OR)  100% estimates vaccine protection. If ‘‘signals’’ of inadequate protection emerge from such screening studies, these studies can then be followed up by more rigorous cohort or case-control studies of vaccine protection. An interesting hybrid design was developed to assess pneumococcal polysaccharide vaccine protection (104). In this design, isolates from cases of invasive pneumococcal disease sent to a referral center were typed and histories of antecedent vaccination were obtained without knowledge of these types. Since conventional pneumococcal polysaccharide vaccine contains only a fraction of pneumococcal serotypes encountered in infected patients and since serotypes contained in the vaccine are not expected to protect against infections caused by other serotypes, the nonvaccine serotype infections can be considered a suitable control group for the vaccine serotype ‘‘cases,’’ and the expression (1
OR)  100% estimates vaccine protection. Finally, because it is sometimes necessary to evaluate putative vaccine side effects in situations in which vaccine

coverage is very high, leaving very few unvaccinated subjects for comparison, an innovative design, termed ‘‘case-series,’’ has been used. In this design, the incidence of the adverse event of interest is compared within two windows of time for the same subject: a ‘‘vaccine window,’’ an interval after dosing during which vaccine-related side effects are postulated to occur; and a ‘‘control window,’’ an interval proximate to dosing during which vaccine-related side effects are postulated not to occur. The ratio of these two incidence rate estimates is taken to estimate the relative rate and/or risk of the event in vaccinees relative to nonvaccinees. This is analogous to crossover designs used in RCTs, in which the essential comparison is the withinsubject occurrence of the target outcome before and after the crossover from one agent to another. This design, which can be used to study acute, transient adverse effects that occur during a predictable time window shortly after vaccination, was used very successfully in the evaluation of intussusception following oral receipt of live rhesus rotavirus reassortant vaccine and produced estimates of the relative risk of this outcome in vaccinees versus nonvaccinees that were quite similar to estimates from case-control studies (105).

Increasing Importance of Population-Based Databases Recent years have witnessed an explosion of allegations about putative serious side effects associated with receipt of vaccines. Some of these alleged associations, such as the occurrence of intussusception following oral receipt of live rhesus rotavirus reassortant vaccine, have been verified by credible scientific studies (105,106). Others, such as the alleged occurrence of inflammatory bowel disease or autism following MMR vaccine, have not been substantiated (107,108). Because assertions about vaccine safety can threaten public confidence in vaccines used in routine practice, it has been proved essential that credible, suitably controlled studies be completed rapidly when such assertions arise. In the past, public health systems relied principally on side effects voluntarily reported by individual physicians. An example of such a system is the Vaccine Adverse Event Reporting System (VAERS) managed by the U.S. Public Health Service (109). Because of the selective and incomplete reporting of side effects inherent in these systems, as well as uncertainties about the denominators of vaccinees at risk and the occurrence of target side effects in nonvaccinees, special large-linked, computerized databases have been created, such as the Vaccine Safety Datalink (VSD) created by the U.S. Centers for Disease Control (110). These databases link histories of receipt or nonreceipt of vaccines in a defined population with comprehensive records of treatment encounters and hospitalizations for specific outcome conditions in the same population. In addition, information about demographic and socioeconomic variables for each subject is collected to permit control for possible confounders in analyses of vaccineadverse event associations. If maintained for a suitably large cohort of the target population, these databases enable rapid, controlled analyses and provide the public health community with the evidence necessary for proper regulation of the usage of licensed vaccines. Unfortunately, although such databases are becoming increasingly common in industrialized countries, they are virtually nonexistent in developing countries (111). One large-linked, dynamic database to evaluate vaccine safety issues has been piloted in Vietnam, but there is a pressing need to develop more such systems in the developing world (112).

Chapter 5: Long-Term Evaluation of Vaccine Performance

Methodological Limitations of Observational Vaccine Evaluations Post-licensure observational studies are indispensable tools for providing information about the safety of vaccines and the protection conferred by them as they are routinely given in practice. Nevertheless, it is important that investigators be aware of certain general limitations of these studies. As already mentioned, the absence of concurrent controls and the indirect focus on populations rather than a direct focus on individuals constitute weaknesses of indirect before-and-after studies, which can severely limit the inferences that can be drawn from these studies. However, even direct study designs can be limited by problems with the quality of information used, by bias, and by intrinsic difficulties in estimating the total protective impact on the target disease, including both direct and indirect protection. Most observational assessments of vaccines rely on retrospective information, either documented or recalled, to ascertain histories of vaccination and the occurrence of disease outcomes. The accuracy of this information may be limited, sometimes significantly, by imperfect recall, inaccurate routine diagnoses, inaccurate records, and incomplete records. These issues are not major problems for RCTs of vaccines, since vaccination is assigned in such trials and surveillance for disease outcomes is prospective, usually incorporating uniform diagnostic procedures and criteria as well as systematic recording of outcomes. Bias in observational studies, which distorts the relationship between vaccination and disease outcomes, can arise either because of the way that a vaccine is given and outcomes are detected in practice or because of the way that investigators choose groups for comparisons and collect information about individuals in these groups. Bias can arise when persons who are vaccinated in practice do not represent the target population for vaccination with respect to their risks of disease outcomes and when vaccinated and nonvaccinated persons do not receive equally intense and accurate diagnostic surveillance for disease outcomes. Moreover, in selecting vaccinees and nonvaccinees for a controlled cohort study or cases and controls for a case-control study, there is the potential for biased choices to be made that can alter vaccine–disease outcome relationships. Finally, biases can occur in classifying the vaccination and disease status of study subjects if decisions about vaccination status are not made without knowledge of disease status and vice versa. As noted earlier, the randomization, double blinding, comprehensive follow-up, and systematic and accurate diagnostic procedures employed in a RCT of a vaccine provide the best available safeguards against these biases. For infections transmitted from person to person, attainment of a sufficiently high level of vaccine coverage of the target population has the potential not only to reduce the susceptibility to infection directly, via induction of protective immunity, but also to augment this protection indirectly by interrupting transmission of the pathogen in the community. However, because post-licensure observational assessments typically compare disease outcomes among vaccinees versus nonvaccinees sampled from the same population and because the benefits of reduced transmission within a population are shared by both vaccinees and nonvaccinees, these studies are poorly designed to measure the total protection of vaccinees and the indirect protection of nonvaccinees that can result from vaccine herd protection (25–27). Finally, despite past successes of the case-series design in evaluating the associations between vaccines and side effects, it

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must be realized that use of the design is limited to the relatively uncommon situations in which clear-cut windows of vulnerability and invulnerability to the study outcome can be defined in advance of the study. Moreover, because the serial order of windows of vulnerability and invulnerability are typically defined in the same way for each subject in a caseseries study and is not randomized as in a crossover clinical trial, these studies are potentially vulnerable to biases that can occur if the incidence of side effects is confounded by ordered effects. Citation of these limitations is not meant to denigrate the value or utility of post-licensure observational studies. Nor is it meant to detract from available tactics in the design, execution, and analysis of these studies that can minimize these potential problems (113). However, it should be clear from this discussion that the double-blinded RCT serves as the gold standard design for ensuring the validity of a vaccine evaluation and that postlicensure observational assessments can only strive to approach the assurance of validity provided by properly done trials.

COMMENT In this chapter, we have attempted to outline the basic paradigm for the design of modern field trials to evaluate new vaccines. The broad outlines of the RCT paradigm are relatively simple and in principle are identical with well-established guidelines for designing trials of clinical therapies (94). Despite this apparent overall simplicity, however, numerous subtle issues arise when the general principles are applied to the specific questions raised in a particular trial. The perspective of the research question must be clarified, and the design of the trial must be carefully adapted so that the study population, compared agents, and outcomes address this question. Wherever possible, strategies must be formulated to minimize the opportunity for distortion of results by bias while, at the same time, yielding estimates of vaccine protection, safety, and immunogenicity with acceptable statistical precision and generalizability. Extensive background data are required to plan a trial with these features, and great care is required to document the performance of the trial and the absence of bias at every stage of its execution. We have not included significant discussion of the additional complexities that occur when RCTs are conducted in a multicenter fashion or in less developed settings, nor have we addressed the many issues that are important in executing a field trial (114). A minimum list of these issues would include scrupulous attention to fulfilling contemporary ethical requirements, which are rapidly changing (114–117); the development and maintenance of productive relationships between the research project and the participating community as well as between scientific collaborators in the project; recruitment, training, and supervision of personnel; procurement and maintenance of supplies and equipment; supervision, coordination, and quality control of field and laboratory procedures; and proper collection and management of data. Even with an excellently designed study, suitable attention to these issues will be required for achievement of a successful trial. Finally, we have attempted to underscore the importance of continued evaluation of the performance of a vaccine, even after it has been deployed in practice. Because such assessments must usually rely on observational study designs, they will be considerably more vulnerable to biases than phase III

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RCTs. Nevertheless, when conducted with attention to safeguards against these biases, such studies provide critical information to public health workers in their attempt to ensure not merely that vaccines are delivered to a high fraction of the targeted population but also that recipients of the vaccine are being protected as expected and are not experiencing unacceptable vaccine side effects.

REFERENCES 1. Deen J, Clemens J. Issues in the design and implementation of vaccine trials in developing countries. Nat Rev Drug Discov 2006; 5:932–940. 2. Lepow M, Nankervis G. Eight-year serologic evaluation of Edmonston live measles vaccine. J Pediatr 1969; 75:407–411. 3. Dam HG, Toman K, Hitze KL, et al. Present knowledge of immunization against tuberculosis. Bull World Health Organ 1976; 54:255–269. 4. Murphy T. Haemophilus b polysaccharide vaccine: need for continuing assessment. Pediatr Infect Dis J 1987; 6:701–703. 5. Shapiro ED, Berg AT, Austrian R, et al. The protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N Engl J Med 1991; 325:1453–1460. 6. Orenstein WA, Bernier RH, Dondero TJ, et al. Field evaluation of vaccine efficacy. Bull World Health Organ 1985; 63:1055–1068. 7. Acosta CJ, Galindo CM, Ali M, et al. A multi-country cluster randomized controlled effectiveness evaluation to accelerate the introduction of Vi polysaccharide typhoid vaccine in developing countries. Trop Med Int Health 2005; 10:1219–1228. 8. Byar DP, Simon RM, Friedewald WT, et al. Randomized clinical trials: perspectives on some recent ideas. N Engl J Med 1976; 295:74–80. 9. Bailar J. Introduction. In: Shapiro SH, Louis TA, eds. Clinical Trials: Issues and Approaches. New York: Marcel Dekker, 1983:1. 10. Greenwood M, Yule G. The statistics of anti-typhoid and anticholera inoculations. Proc R Soc Med 1915; 8:113. 11. Szmuness W, Stevens CE, Harley EJ, et al. Hepatitis B vaccine: demonstration of efficacy in a controlled clinical trial in a high risk population in the United States. N Engl J Med 1980; 303: 833–841. 12. Institute of Medicine. New vaccine development. Establishing Priorities: Vol 2. Diseases of Importance in Developing Countries. Washington, DC: National Academy Press, 1986:241. 13. Levine RJ, Lebacqz K. Ethical considerations in clinical trials. Clin Pharm Ther 1979; 25:728–749. 14. Clemens J, Horwitz R. Longitudinal methods for evaluating therapy. Biomed Pharmacother 1984; 38:440–443. 15. Feinstein AR. Clinical biostatistics—III: the architecture of clinical research. Clin Pharmacol Ther 1970; 11:432–441. 16. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient: I. Introduction and design. Br J Cancer 1976; 34:585–612. 17. Peto R, Pike MC, Armitage P, et al. Design and analysis of randomized clinical trials requiring prolonged observation of each patient: II. Analysis and examples. Br J Cancer 1977; 35:1–39. 18. Chalmers TC. The control of bias in clinical trials. In: Shapiro SH, Louis TA, eds. Clinical Trials: Issues and Approaches. New York, Marcel Dekker, 1983:115. 19. Feinstein AR. Clinical biostatistics: XXII. The role of randomization in sampling, testing, allocation, and credulous idolatry (part 1). Clin Pharmacol Ther 1973; 14:601–615. 20. Feinstein AR. Clinical biostatistics: XXIII. The role of randomization in sampling, testing, allocation, and credulous idolatry (part 2). Clin Pharmacol Ther 1973; 14:898–915. 21. Feinstein AR. Clinical biostatistics: XXIV. The role of randomization in sampling, testing, allocation, and credulous idolatry (conclusion). Clin Pharmacol Ther 1973; 14:1035–1051.

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6 Ethical Considerations in the Conduct of Vaccine Trials in Developing Countries Charles Weijer Department of Philosophy, University of Western Ontario, London, Ontario, Canada

Claudio F. Lanata Instituto de Investigacio´n Nutricional La Molina, Lima, Peru

Christopher V. Plowe Howard Hughes Medical Institute and Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION In the last three decades of the ethics of research, no single issue has evoked as much controversy as the conduct of clinical trials in developing countries. Facets of the controversy include the questions: Is there a single international standard for informed consent? Is there a role for community consultation and consent? Where should clinical trials be conducted first? Which standard of care, that of the host or that of the sponsor country, is the right standard? What other medical care must be provided to research subjects? What is owed to research participants at the conclusion of the study? How to manage review of research by multiple ethics committees? Each of these questions emanates ultimately from global cultural diversity and global inequities in health. A debate was sparked by a 1997 article in the New England Journal of Medicine challenging the use of placebo in clinical trials testing short-course zidovudine for the prevention of perinatal transmission of HIV (1). At the time of the trials, the AIDS Clinical Trial Group (ACTG) 076 regimen was widely used in developed countries and was known to be effective in reducing the rate of perinatal transmission of HIV by approximately two-thirds. The authors argued that the use of placebo rather than the ACTG 076 regimen as a control treatment in these trials deprived research participants access to effective treatment. In effect, the trials were thought to reflect a double standard for research in developed and developing countries. Defenders of the short-course zidovudine trials pointed out that the call for these trials came from developing countries themselves and that important differences relevant to perinatal transmission of HIV existed between developing and developed countries: antenatal care was not widely accessible in developing countries; facilities for the intravenous administration of zidovudine required by the ACTG 076 regimen did not exist; mothers infected with HIV were nonetheless being advised to breast feed; and the ACTG 076 regimen was simply unaffordable (2). Thus, the relevant question to physicians and their patients in developing countries was not how the shortcourse regimen compares to an inaccessible treatment, rather

how it compared to best available care locally (3). Such differences in priorities and circumstances between developed and developing nations must also be considered in the case of vaccine trials. Vaccine research in developing countries has not been immune to controversy. A series of clinical trials demonstrated that the tetravalent rhesus rotavirus (RRV-TV) vaccine prevented serious rotavirus diarrhea in developed countries, (4) as well as in developing countries (5). After being approved by the U.S. Food and Drug Administration, it was recommended for universal use in the United States. Once widespread vaccination in the United States was underway, and 1.5 million doses were given, however, a transient association between RRV-TV vaccination and intussusception was observed. In October 1999, while the definitive studies to prove this association were under way, the vaccine manufacturer voluntarily withdrew the RRV-TV vaccine from the U.S. market. This led to the dispute as to whether research with the vaccine should (or could) continue in developing countries (6). Even though many considered the much greater benefit of rotavirus vaccines in developing countries to outweigh the risks, concerns about a double standard (or appearances thereof) effectively ended further testing. In the wake of the controversy, developing countries would have to wait another seven years for new rotavirus vaccines to be developed (7). In 2004, international controversy erupted over a series of HIV prevention trials using an antiretroviral drug (tenofovir), involving a variety of high-risk groups in developing countries (8). Trial participants, who would receive risk-reduction counseling, male condoms, and treatment for other sexually transmitted diseases, were to be randomized to tenofovir or placebo in the hope that the drug would reduce HIV infection rates. Just as the Cambodian clinical trial was to get underway in 2004, sex workers calling themselves the ‘‘Asian Pacific Network of Sex Workers’’ and AIDS Coalition to Unleash Power (ACT UP) Paris, a French HIV/AIDS activist group, disrupted the International AIDS Conference in Bangkok, Thailand. Given that a substantial proportion of the subjects

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would likely contract HIV during the study, protesters claimed that the failure to provide lifelong treatment for HIV to infected research subjects is immoral. As a consequence, the trials were suspended internationally. The events surrounding the tenofovir trials led to an international debate regarding moral obligations to subjects who become infected during prevention trials. The purpose of this chapter is to provide the vaccine researcher working in developing countries with an understanding of the basic ethical principles of research, international regulations, and current ethical controversies when conducting vaccine research with human subjects in developing countries.

ETHICAL PRINCIPLES The conduct of clinical research is guided by four ethical principles: respect for persons, beneficence, justice, and respect for communities (Table 1). The principle of respect for persons requires that researchers take the choices of people who are capable of deciding for themselves seriously. Furthermore, those who cannot decide for themselves are entitled to protection. This principle maps onto the moral rules of informed consent and confidentiality. The investigator is obliged to obtain agreement from each research subject for study participation. For informed consent to be valid, the research subject must have the capacity to make the choice, be able to make a free choice, be adequately informed, and understand the information with which he or she has been presented. The investigator must also take necessary steps to protect the confidentiality of the research subject’s health information. The principle of beneficence requires that investigators not harm and, where possible, promote the welfare of research subjects. The complexity of risk analysis in research belies simplistic expressions, such as an ‘‘acceptable benefit-harm ratio’’ or ‘‘balance of benefits to harms.’’ The first step in unpacking these metaphors is the recognition that clinical research may contain a mixture of procedures, some offering potential benefit to research subjects (therapeutic procedures), while others are administered solely to answer the study question (nontherapeutic procedures) (9). Therapeutic procedures in clinical research are justified if they satisfy clinical equipoise. This means they must be roughly Table 1 Ethical Principles and Rules Governing the Conduct of Human Subjects Research Moral principle

Moral rule

Respect for persons

Obtain the informed consent of prospective research subjects. Protect the confidentiality of private information. Therapeutic procedures must satisfy clinical equipoise. Risks of nontherapeutic procedures must be (i) minimized and (ii) reasonable in relation to knowledge to be gained. Subject selection procedures must be fair. Compensate subjects harmed as a result of research participation. Respect communal values, protect and empower social institutions. Where applicable, abide by the decisions of legitimate communal authority.

Beneficence

Justice

Respect for communities

comparable with competent medical care. Formally, there must be a state of honest, professional disagreement in the community of expert practitioners as to the preferred treatment (10). Nontherapeutic procedures, by definition, do not offer the prospect of benefit to individual study participants, and hence a harm-benefit calculus is inappropriate. Rather, nontherapeutic procedures are acceptable if the risks associated with them are minimized consistent with sound scientific design, and reasonable in relation to the knowledge to be gained (9). For a study to be allowed to proceed, the moral rules for both therapeutic and nontherapeutic procedures must be passed. The principle of justice may be defined as the ethical obligation to treat people fairly. Investigators have an obligation to ensure that subject selection procedures are fair. They must neither exploit the vulnerable, nor exclude without good reason those who stand to benefit from study participation. For proposed eligibility criteria to be evaluated, each criterion must be accompanied by a clear justification in the study protocol. The inclusion of a vulnerable population requires a clear justification. Further, in so far as is possible and practicable, the study population ought to mirror the target clinical population. The principle of justice also requires that provisions be in place to compensate research subjects who are harmed as a result of research participation. A novel ethical principle of respect for communities has been proposed (11). There is much support for the principle. First, the community (or communities) to which we belong is an important source of values and self-understanding. Second, a community consists of social structures that are essential to the well being of its members. Third, the principle acknowledges that some communities already legitimately exercise power to make binding decisions on behalf of members, for instance, in the collection of taxation or the setting of speed limits on roads. The principle of respect for communities implies that investigators have an obligation to respect communal values, protect and empower social institutions, and, where applicable, abide by the decisions of legitimate communal authorities.

INTERNATIONAL REGULATION International codes of ethics for research are a response to a history of abuses in research involving human subjects. During the Nuremberg war crimes trials, an influential set of principles for research was drawn up in 1947, which were later known as the Nuremberg Code. Subsequently, the United Nations General Assembly signed in 1948 the Universal Declaration of Human Rights, which includes a right not to be experimented upon without informed consent. In 1953, the World Medical Association (WMA) began drafting a code specifically for physicians conducting research. It was not, however, until 1964 that the recommendations were adopted as the Declaration of Helsinki. The Declaration of Helsinki has been revised six times, most recently in 2008 (12). The Council for International Organizations of Medical Sciences (CIOMS) published a lengthy and detailed commentary on the Declaration of Helsinki, with a special emphasis on research conducted in developing countries, in 1993, called the International Ethical Guidelines for Biomedical Research Involving Human Subjects. The document underwent a substantial revision in 2002 (13). In 1996, the International Conference on Harmonization (ICH) published its Guideline for Good Clinical Practice. The document provides unified technical standards for clinical trials, so data generated

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Table 2 Chronology of International Ethics Guidelines for Biomedical Research Year

Document

1947 1948 1964 1966 1975 1983 1989 1989 1991 1993

Nuremberg code Universal declaration of human rights Declaration of Helsinki (1) International covenant on civil and political rights Declaration of Helsinki (1st revision—Tokyo) Declaration of Helsinki (2nd revision—Venice) Declaration of Helsinki (3rd revision—Hong Kong) Convention on the rights of children International guidelines for ethical review of epidemiological studies International ethical guidelines for biomedical research involving human subjects Guidelines for good clinical practice for trials on pharmaceutical products Declaration of Helsinki (4th revision—South Africa) ICH Guidance on Good Clinical Practice

1995 1996 1996* 2000 2000 2000 2002 2002 2002a 2004 2007a 2008a

Issuing authority

Declaration of Helsinki (5th revision—Scotland) Ethical considerations in HIV preventive vaccine research Operational guidelines for ethics committees that review biomedical research Declaration of Helsinki (note of clarification on paragraph 29) Surveying and evaluating ethical review practices International ethical guidelines for biomedical research involving human subjects Declaration of Helsinki (note of clarification on paragraph 30) Ethical Considerations in Biomedical HIV Prevention Trials Declaration of Helsinki (6th revision—Seoul)

United Nations General Assembly WMA United Nations General Assembly WMA WMA WMA United Nations General Assembly CIOMS/WHO CIOMS/WHO WHO WMA ICH/Committee for Proprietary Medical Products for the Pharmaceutical Industry WMA UNAIDS WHO WMA WHO CIOMS/WHO WMA UNAIDS WMA

a

The four most-quoted guidelines for the conduct of biomedical research with human subjects in developing countries include the following:

. The Declaration of Helsinki, in its last revision contains a terse articulation of 32 principles to guide the conduct of research. . The Guidance on Good Clinical Practice of the ICH of 1996 provides unified technical standards for clinical trials, so data generated in one country would be mutually acceptable by regulatory authorities the United States, Japan, and the European Union. . The International Ethical Guidelines for Biomedical Research Involving Human Subjects of CIOMS/WHO 2002 are a lengthy and detailed commentary on the Declaration of Helsinki with a special emphasis on research conducted in developing countries. It is intended to help WHO country members to develop their own national ethical policies for clinical research, guiding them how to adapt international ethical principles to their local realities, and to establish adequate procedures for the ethical review of research protocols of studies with human subjects participation. These guidelines contain 23 major recommendations. . Ethical Considerations in Biomedical HIV Prevention Trials of the UNAIDS of 2007, which were created to help the conduct of this type of research as a response to the current controversies. This document contains 19 guidance points and is unique in its focus on international HIV prevention research. Abbreviations: WMA, World Medical Association; CIOMS, Council for International Organizations of Medical Sciences; WHO, World Health Organization; ICH, International Conference on Harmonisation; UNAIDS, Joint United Nations Programme on HIV/AIDS.

in one country would be mutually acceptable by regulatory authorities the United States, Japan, and the European Union (14). In 2000, Joint United Nations Programme on HIV/AIDS (UNAIDS) published Ethical Considerations in HIV Preventive Vaccine Research to provide guidance to HIV vaccine researchers. This document, renamed Ethical Considerations in Biomedical HIV Prevention Trials, was substantially revised in 2007 (15). Clearly, researchers conducting vaccine trials in developing countries face a complex web of international regulations (Table 1). What guidance can be distilled for researchers? The most relevant international documents to vaccine researchers are the WMA Declaration of Helsinki, CIOMS International Ethical Guidelines for Biomedical Research Involving Human Subjects, ICH Guideline for Good Clinical Practice, and the UNAIDS Ethical Considerations in Biomedical HIV Prevention Trials (Table 2). As we shall discuss below, each of the documents contains controversial provisions. The documents also possess important elements in common. As each document is guided by the same moral principles, it is not surprising that there is considerable convergence among these documents. All the documents require that proposals to conduct clinical research be submitted to an independent committee to ensure ethical acceptability (WMA 15; CIOMS 2; ICH 2.6; UNAIDS 4). Informed consent must be

obtained from study participants (WMA 24; CIOMS 4; ICH 2.9; UNAIDS 16). If a potential research subject is incapable of providing consent, then the consent to study participation must be sought from the subject’s legally authorized representative (WMA 28; CIOMS 4; ICH 4.8.5; UNAIDS 10). The potential benefits and harms of study participation must be carefully evaluated (WMA 18; CIOMS 8; ICH 2.2; UNAIDS 11, 12). Finally, vulnerable populations in research are entitled to special protection (WMA 9; CIOMS 13; ICH 3.1.1; UNAIDS 8). While this general guidance is of use to all researchers, those conducting vaccine trials in the international setting require more specific guidance, especially on issues of current controversy. The following guidance is based on provisions in one or more of the relevant international guidelines as interpreted through the lens of grounding moral principles.

CURRENT CONTROVERSIES 1.

Is there one international standard for informed consent? The obligation to obtain informed consent from research participants is well established. Precisely how informed consent is sought may reasonably differ from one context to another. The principle of respect for persons

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requires that researchers be sensitive to beliefs and values of the group to which prospective study participants belong. If, as all the documents require, study participants are to be adequately informed, the informed consent process must ensure that details of the research project are expressed in a way that is locally comprehensible. Thus, the disclosure process must take into account local beliefs, literacy, and education. Must research subjects sign a consent document? This concern flows, in part, from the requirement in U.S. regulation to document the consent process. From a moral rather than regulatory perspective, however, what matters is the quality of the consent process, and not whether a form is signed. In some cultural and political contexts, signing an official form may be associated with different meanings than in the United States, and hence may be an inappropriate requirement. Thus, the CIOMS guidelines observe: ‘‘Consent may be indicated in a number of ways. The subject may imply consent by his or her voluntary actions, express consent orally, or sign a consent form’’ (CIOMS 4). In other cases, documentation of consent may pose a substantial risk to subjects if their medical condition is stigmatized. Thus, a waiver of documentation of consent ‘‘may also be approved when existence of a signed consent form would be an unjustified threat to the subjects’ confidentiality’’ (CIOMS 4). Before these exceptions are invoked, however, locally acceptable ways of documenting the consent process should be explored. Careful consultation with the community may yield acceptable strategies for removing barriers to written informed consent (16). Is there a role for community consultation and consent? Historical approaches to research ethics have been criticized for being unduly individualistic, and failing to take into account the interests of communities (17). As a result, a novel principle of respect for communities has been proposed. The new principle’s implementation, however, poses difficult challenges for the researcher working with communities. The CIOMS guidelines require that research be ‘‘responsive to the health needs and the priorities of the population or community in which it is to be carried out’’ (CIOMS 10). Ensuring that research is responsive to a particular community’s health needs requires dialogue between community and researcher. Thus, ‘‘trial sponsors should consult communities through a transparent and meaningful participatory process which involves them in an early and sustained manner in the design, development, implementation, monitoring, and distribution of results of . . . trials’’ (UNAIDS 2). In some communities, for instance aboriginal communities, the burden of decision making may rest traditionally more with community leaders than individual community members (17). As the CIOMS guidelines point out: ‘‘In some cultures or groups, a researcher may enter a community to conduct research or approach prospective subjects for their individual consent only after obtaining permission from a community leader, a council of elders, or other designated authority. Such customs must be respected’’ (CIOMS 4). The moral obligation to show respect for communities must be tempered with the simultaneous duty to demonstrate respect for persons. Community consent and individual consent have an asymmetrical relationship. The community’s refusal may preclude a researcher’s ability to approach community

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members for consent. ‘‘In no case, however, may the permission of a community leader or other authority substitute for individual informed consent’’ (CIOMS 4). In practice, stepwise processes for both community ‘‘permission to enter’’ and individual informed consent can be developed through consultation with local communities (16). Where should clinical trials first be conducted? For the outsider, one of the curious things about the rotavirus vaccine story, described above, is the fact that clinical trials of the vaccine were first conducted in developed countries, while the majority of mortality from the disease occurs in developing countries. A policy decision to test vaccines of interest to developing countries first in developed countries will predictably lead to two consequences (6). First, the adoption of useful vaccines in developing countries will be delayed as testing is first done elsewhere. Second, the practice has the unintended effect of setting the bar for adoption of a vaccine too high. A country with a low burden of disease will (appropriately) be less likely to accept even small risks associated with a vaccine than a country with a high burden of disease. Thus, any absolute requirement to test a new vaccine in developed countries before testing in developing countries will impede vaccine development. In other areas of clinical research, it is generally accepted that clinical trials ought to be first conducted in high-risk populations, for if a new treatment fails, it is unlikely to be of use to any population. The same follows for vaccine clinical trials. It is important to recognize that there is no insuperable ethical obstacle to conducting early clinical trials in developing countries. The statement found in the UNAIDS document might well be generalized to all vaccine clinical trials. ‘‘Generally, earlier clinical phases of HIV vaccine research should be conducted in communities that are less vulnerable to harm or exploitation, usually within the sponsor country. However, countries may choose, for valid scientific and public health reasons, to conduct any study phase within their populations, if they are able to ensure sufficient scientific infrastructure and sufficient ethical safeguards’’ (UNAIDS 5). Differential burdens of disease or important biological differences between developed and developing country would satisfy the requirement for ‘‘valid scientific and public health reasons’’. As scientists and ethical review boards in developing countries gain experience with testing vaccines and establish the capacity to manage serious unexpected adverse events, they may become more comfortable with the idea of conducting the first trials of vaccines. Which standard of care, of the host or sponsor country, is the right standard? The perinatal HIV prevention trials, described above, highlight a deep divide in research ethics regarding the nature of the researcher’s obligation to research subjects. A core norm in research ethics is that the medical care of the research subject ought not be disadvantaged by study participation. In the Declaration of Helsinki, this norm is expressed as follows: ‘‘The benefits, risks, burdens and effectiveness of a new intervention must be tested against those of the best current proven intervention’’ (WMA 32). As there are inequities globally in the distribution of health care resources, one might well ask: ‘‘[T]he best current . . . intervention’’ where?

Chapter 6: Ethical Considerations in the Conduct of Vaccine Trials

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One might answer that the requisite standard should be that in a developed country or, alternatively, one might answer that it ought to be that in a developing country. Each possibility has its proponents; each can point to an international ethics document to support its case. While the text of the Declaration of Helsinki is ambiguous on this issue, the intention of its authors is not. In a press release, the WMA states: ‘‘The WMA opposes the notion that the nonavailability of drugs should be used as a justification to conduct placebo-controlled trials. Dr Human [former WMA Secretary General] said that ‘this would lead to poor countries of the world being used as the laboratory of research institutions of the developed world’ ’’ (18). Adopting a local standard of care in isolation surely would present just such a risk to developing countries. No one to our knowledge is, however, suggesting such a move. Proponents of a local standard of care point out that other protections will prevent exploitation. Central to these protections is the requirement that research be ‘‘responsive to the health needs and the priorities of the population or community in which it is to be carried out’’ (CIOMS 10). It is difficult to imagine an exploitive study that would both pass a local standard of care threshold and meet the health needs and priorities of the community in the developing country. Furthermore, the adoption of a local standard of care threshold also must not be allowed to take advantage of inefficiencies in a developing country’s health care system. One may morally distinguish between the stated policies and objectives of a health care system (de jure local standard of care) and its implementation in the field (de facto local standard of care). The former, and not the latter, should guide the choice of a control treatment for a clinical trial (19). Thus, in a country that has an imperfectly implemented policy of universal provision of a vaccine, that vaccine should not be withheld from the control group in a trial of a new vaccine for that same indication. What other medical care must be provided to research subjects? The controversy surrounding the tenofovir HIV prevention trials precipitated an international debate as to ethical obligations to research subjects infected with HIV during the course of a prevention trial. The ethical principle of justice grounds the obligation to compensate subjects for research-related injury. This obligation is reflected in the CIOMS document, ‘‘Investigators should ensure that research subjects who suffer injury as a result of their participation are entitled to free medical treatment for such injury and to such financial or other assistance as would compensate them equitably for any resultant impairment, disability or handicap’’ (CIOMS 19). Just what constitutes a research-related injury in the context of a prevention trial requires careful consideration. Childress, in his seminal paper on the topic, uses a positional-risk test: ‘‘It asks whether the injury would have been avoided if the injured party had not been in that position (i.e., a research participant)’’ (20). The UNAIDS document correctly traces out the implications of this standard for HIV vaccine studies: ‘‘HIV infection acquired during participation in a biomedical HIV prevention trial should not be considered a compensable injury unless directly attributable to the prevention product being tested itself, or to direct contamination through a research-related activity’’ (UNAIDS 9). Thus, the development of infection because of

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risk behaviors of the research subject is not a researchrelated injury for which treatment must be provided. The UNAIDS document goes on to identify an unprecedented obligation to treat all infection in prevention trials, even infection that is not a research-related injury. It states: ‘‘Participants who acquire HIV infection during the conduct of a biomedical HIV prevention trial should be provided access to treatment regimens from among those internationally recognized as optimal’’ (UNAIDS 14). The document cites ethical principles of beneficence and justice as the foundation of this obligation, despite the existence of an unanswered refutation of the claim (21). We are concerned that the widespread adoption of this purported obligation would have a chilling effect on international vaccine research. What is owed to research participants at the conclusion of the study? A further protection for research subjects and the communities in which they live is afforded by the obligation to share research benefits with study participants. The precise scope of this obligation is, however, a matter of controversy. In the very least, researchers have an obligation to persons who actually participated in the trial. Thus, the Declaration of Helsinki requires that, ‘‘At the conclusion of the study, patients entered into the study are entitled . . . to share any benefits that result from it, for example, access to interventions identified as beneficial in the study. . .’’ (WMA 33). But might obligation be reasonably construed as broader than this? Some have argued that it may be. The CIOMS document broadens the scope of this requirement considerably in its position that the researcher and sponsor have an obligation to ensure that ‘‘any intervention or product developed, or knowledge generated, will be made reasonably available for the benefit of that population or community’’ (CIOMS 10). The CIOMS document takes the further step in suggesting that researchers and sponsors have an obligation to ‘‘see that biomedical research projects for which they are responsible in such countries contribute effectively to national or local capacity to design and conduct biomedical research, and to provide scientific and ethical review and monitoring of such research’’ (CIOMS 20). The UNAIDS document requires that trial sponsors and developing countries come to an agreement on posttrial access to treatment. It states, ‘‘[T]rial sponsors and countries should agree on responsibilities and plans to make available as soon as possible any biomedical HIV preventive intervention demonstrated to be safe and effective . . . to all participants in the trials in which it was tested, as well as to other populations at higher risk of HIV exposure in the country’’ (UNAIDS 19). The document goes on to claim that ‘‘making a successful HIV biomedical HIV prevention product or intervention reasonably available to the population where it was tested can be sustained as a basic ethical requirement’’ (UNAIDS 19). The feasibility of these recommendations for the provision of treatment to entire community, population, or country may be questioned. For instance, it would have been very difficult, or even economically impossible, after completing the rotavirus vaccine trials in periurban Lima, where 800 infants participated, to provide the vaccine broadly (22). The cost of the vaccine when introduced in the U.S. market was US$27 per dose (23). If the scope of the

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obligation were limited to infants in Lima (the community), this would require the vaccination, for free or at a substantially reduced price, of 150,000 children each year at a cost of US$12,000,000. If the scope were broader yet (the country), it would require the vaccination of the 600,000 infants that are born each year in the whole of Peru at a cost of US$48,000,000. And then ask for how long: one year, five years, or in perpetuity? Clearly, the obligation cannot be interpreted so broadly as to render impossible clinical trials designed to develop important vaccines for developing countries. How to manage review of research by multiple ethics committees? The review of research by multiple ethics committees poses challenges to all researchers conducting multiinstitutional research. Submissions to multiple committees result inevitably in lengthy delays. Worse yet, investigators are likely to encounter divergent responses from differing committees, and required changes from one committee may contradict required changes from another. As difficult as these problems are, they are commonplace and well recognized. Researchers conducting vaccine trials in developing countries face what we believe is an even more difficult problem when research must be approved by ethics committees in both host and sponsor country. The commentary on CIOMS guideline 22 states, ‘‘Committees in both the country of the sponsoring agency and the host country have responsibility for conducting both scientific and ethical review, as well as the authority to withhold approval of research proposals that fail to meet their scientific or ethical standards’’ (CIOMS 22). Conflict predictably ensues from the question: Whose ethical standards shall be followed? The U.S. government now requires that any foreign institution conducting U.S. funded research sign a document affirming that it will comply with U.S. ethics standards (Assurance of Compliance). One result of this is that rigid criteria are applied to the consent forms used in developing countries. As a result, the length and complexity of information in consent forms has significantly increased over time, perhaps making it more difficult for prospective subjects to understand. When efforts are undertaken in the host country to simplify forms, ethics committees in the sponsoring developed countries often resist even the most reasonable changes. Two solutions to this situation present themselves. First, the CIOMS guidelines allow for a separation of activities between ethics committees; even though responsibility remains joint. CIOMS says, ‘‘When a sponsor or researcher in one country proposes to carry out research in another country, the ethical review committees in the two countries may, by agreement, undertake to review different aspects of the research protocol . . .’’ (CIOMS 22). Thus, by agreement, the ethics committee in the sponsoring country could have responsibility to assure the adequacy of the proposed scientific methods and the study’s capacity to answer the objectives of the study. The ethics committee in the host country, on the other hand, could then focus on ensuring that the study responds to the needs of the country, selection of study participants is equitable, and informed consent will be obtained appropriately. Second, the United States must utilize the so-called protections equivalent clause. Research funded by the

U.S. government generally must abide by provisions set out in the U.S. federal Common Rule, as well as applicable international regulation (24). Recognizing that international standards may not be identical to U.S. regulations, the Common Rule has a provision allowing for other regulations to be followed. It states that [if] a Department or Agency head determines that the procedures prescribed by the institution afford protections that are at least equivalent to those provided in this policy, the Department or Agency head may approve the substitution of the foreign procedures in lieu of the procedural requirements provided in this policy [45 CFR 46.101(h)]. U.S. regulators have yet to specify exactly which international regulations fulfill the ‘‘protections at least equivalent’’ clause, thereby limiting its application. In practice, while it is unusual for either developed or developing country ethics committees to give up responsibility for reviewing of major aspects of research protocols to an overseas committee, we are aware of occasional examples when one U.S. Institutional Review Board (IRB) has deferred review of a protocol that is also reviewed by another U.S. IRB and an ethical review committee in Africa. When review committees disagree, some have advocated giving jurisdictional preference to qualified local review committees, arguing that these are the best informed of local conditions, and best able to assess risks and benefits (25). Even such small matters as which committee letterhead the consent forms are printed on may require negotiation and compromise. We suggest that in general, the local committee should prevail.

CONCLUSION There can be little question that many challenges face the vaccine researcher in planning and conducting research in a developing country. The task is made more difficult by divergence among ethics guidelines. We hope that future revisions of current guidelines will provide guidance on current controversies and will aim to minimize divergence among documents. It is encouraging that separate bodies in developed countries, like the U.S. National Bioethics Advisory Commission or the Nuffield Council on Bioethics in the U.K., have indicated that solution to these controversies are not to be found in increased regulations, but in closer ties with developing countries and their institutions and researchers (26). We agree with this approach. Dialogue and investment are required to join the divide between developed and developing countries in the context of research. There is an urgent need for investment in local capacity for research in developing countries. Only with funds and training from developed countries will researchers in developing countries be able to design and implement their own clinical trials addressing local health priorities. Investment is also required in the development of infrastructure and training for the ethical review of research. The Nuffield Council on Bioethics correctly observes, ‘‘the guidelines and the Declaration [of Helsinki] will not be effective unless they are accompanied by training and the necessary resources to allow its adequate implementation in developing countries’’ (27).

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REFERENCES 1. Lurie P, Wolfe SM. Unethical trials of interventions to reduce perinatal transmission of the human immunodeficiency virus in developing countries. N Engl J Med 1997; 337:853–856. 2. Varmus H, Satcher D. Ethical complexities of conducting research in developing countries. N Engl J Med 1997; 337:1003–1005. 3. Crouch RA, Arras JD. AZT trials and tribulations. Hastings Cent Rep 1998; 28(6):26–34. 4. Rennels MB, Glass RI, Dennehy PH, et al. Safety and efficacy of high-dose rhesus-human reassortant rotavirus vaccines—report of the National Multicenter Trial. Pediatrics 1996; 97:7–13. 5. Linhares AC, Lanata CF, Hausdorff WP, et al. Reappraisal of the Peruvian and Brazilian lower titer tetravalent rhesus-human reassortant rotavirus vaccine efficacy trials: analysis by severity of diarrhea. Pediatr Infect Dis J 1999; 18:1001–1006. 6. Weijer C. The future of research into rotavirus vaccine. Br Med J 2000; 321:525–526. 7. Glass RI, Parashar UD. The promise of new rotavirus vaccines. N Engl J Med 2006; 354:76–77. 8. Anonymous, The trials of tenofovir trials. Lancet 2005; 365:1111. 9. Weijer C, Miller PB. When are research risks reasonable in relation to anticipated benefits? Nat Med 2004; 10:570–573. 10. Freedman B. Equipoise and the ethics of clinical research. N Engl J Med 1987; 317:141–145. 11. Weijer C, Emanuel EJ. Protecting communities in biomedical research. Science 2000; 289:1142–1144. 12. World Medical Association. Declaration of Helsinki, 2008. Available at: http://www.wma.net/e/policy/b3.htm. Accessed August 30, 2009. 13. Council for International Organizations of Medical Sciences (CIOMS). International Ethical Guidelines for Biomedical Research Involving Human Subjects. Geneva: CIOMS, 2002. Available at: http://www.cioms.ch/frame_guidelines_nov_2002.htm. Accessed December 15, 2007. 14. International Conference of Harmonization. Guideline for Good Clinical Practice. Available at: http://www.ich.org/LOB/media/ MEDIA482.pdf. Accessed December 23, 2007. 15. Joint United Nations Programme on HIV/AIDS (UNAIDS). Ethical considerations in HIV preventive vaccine research. Geneva: UNAIDS, 2000. Available at: http://data.unaids.org/Publications/ IRC-pub01/jc072-ethicalcons_en.pdf. Accessed December 15, 2007.

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16. Diallo DA, Doumbo OK, Plowe CV, et al. Community permission for medical research in developing countries. Clin Infect Dis 2005; 41:255–259. 17. Weijer C, Goldsand G, Emanuel EJ. Protecting communities in research: current guidelines and limits of extrapolation. Nat Genet 1999; 23:275–280. 18. World Medical Association. Media Release, When are placebo controlled trials ethically acceptable? November 28, 2001. Available at: http://www.wma.net/e/press/2001_1.htm. Accessed December 15, 2007. 19. London AJ. The ambiguity and the exigency: clarifying ‘‘standard of care’’ arguments in international research. J Med Philos 2000; 25:379–397. 20. Childress JF. Compensating injured research subjects: the moral argument. Hastings Cent Rep 1976; 6(6):21–27. 21. Weijer C, LeBlanc GJ. Balm of Gilead: is the provision of treatment to those who seroconvert in HIV prevention trials a matter of moral obligation or moral negotiation? J Law Med Ethics 2006; 34:793–808. 22. Lanata CF, Midthun K, Black RE, et al. Safety, immunogenicity and protective efficacy of one or three doses of the Rhesus tetravalent rotavirus vaccine in infants from Lima, Peru. J Infect Dis 1996; 174:268–275. 23. Ehrenkranz P, Lanata CF, Penny ME, et al. A policy decision for Peru: Should rotavirus vaccine be included in the national immunization program? Rev Panam Salud Publica 2001; 10:240–248. 24. Department of Health and Human Services. Protection of human subjects. 45 Code of Federal Regulations 46, 2005. Available at: http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46. htm. Accessed December 15, 2007. 25. Mfutso-Bengu JM, Taylor TE. Ethical jurisdictions in biomedical research. Trends Parasitol 2002; 18:231–234. 26. National Bioethics Advisory Commission. Ethical and policy issues in international research: clinical trials in developing countries. 2001. Available at: http://www.bioethics.gov/reports/ past_commissions/nbac_international.pdf. Accessed December 15, 2007. 27. Nuffield Council on Bioethics. Report: The ethics of research related to healthcare in developing countries. 2002. Available at: http://www.nuffieldbioethics.org/go/ourwork/developingcountries/publication_309.html. Accessed December 15, 2007.

7 Vaccine Economics: Assuring That Vaccines Are Developed for and Available in Developing Countries Amie Batson Health, Nutrition, Population, Human Development Network, The World Bank, Washington, D.C., U.S.A.

Piers Whitehead Corporate Development, Neovacs SA, Paris, France

INTRODUCTION: THE IMPORTANCE OF ECONOMICS The development, commercialization, and broad availability of vaccines depend not only on the scientific knowledge underpinning a candidate but also on the economic costs, risks, and returns associated with the required investments. Ultimately, assuring the best use of limited resources, or economics, is an important factor in all vaccine decisions, whether it is which disease to target, which candidates to develop, how much capacity to build, or what markets to target at what prices. Understanding the economics that guides these industry decisions will help ensure more efficient and effective strategies that achieve the objectives of accelerating the development and broad availability of priority vaccines. In this chapter, we discuss the role economics plays in vaccine development and commercialization, with a focus on the developing world. We begin by describing the characteristics of the vaccine market and the costs to develop and produce a product. Next, we explore the economic bottlenecks impacting investment in vaccines. We briefly discuss the options for addressing the economic challenges, categorized as ‘‘push’’ and ‘‘pull’’ approaches highlighting two mechanisms, the International Finance Facility for Immunization (IFFIm) and a case study of an advanced market commitment (AMC). We conclude by summarizing both the progress that has been made and the challenges that remain in this critical endeavor.

THE MARKET: VACCINE DEMAND, SUPPLY, AND PRICING The Market The global vaccine market is estimated at over $17 billion in 2007, versus a market of $6 billion in 2000 (1). This represents an annual growth rate of over 16%, substantially higher than both the growth in the vaccine market in the 1990s (9%) and the growth of the pharmaceutical market overall.

a

The vast majority of this growth is due to the introduction and increased use of new, relatively high priced, proprietary products in industrial and middle-income markets. These new products include recombinant human papillomavirus vaccines; second-generation rotavirus vaccines; a varicella-zoster vaccine; and the increased market penetration of Wyeth’s Prevnar1 pneumococcal conjugate, which alone added $2 billion in sales between 2000 and 2007 (2,3). There has also been significant growth in international sales to the low-income country market, currently estimated at $350 to $500 million. New money is being invested in immunization for the world’s poorest children, much of it funneled through the Global Alliance for Vaccines and Immunization (GAVI), a public-private partnership established in 2000. GAVI notes that vaccine spending on children in the 72 GAVI-eligible countries (GDP per capita of under $1000/yr) has risen from $2.50 per child in 2000 to $5 per child in 2005, a growth rate of just less than 15% (4). While the market is significant, it should be remembered that while the 72 GAVI-eligible countries have a combined population of 2.8 billion, or 42% of the world population, their sales represent less than 2% to 3% of global revenues.

Vaccine Suppliers The vaccine industry is increasingly concentrated, with five multinationals, GlaxoSmithKline, Sanofi Pasteur, Wyeth, Merck, and Novartis, now accounting for over 90% of industry revenuesa versus these companies or their predecessor entities with 60% in 1988. Industry consolidation has played a role in this increasing concentration, as have companies exiting the vaccine business: Of the 20 internationally active companies in the 1960s, fewer than 12 remained by the early 1990s (5). Most significant, however, has been that these five companies have been largely responsible for the introduction of innovative proprietary products at prices that, relative to historical norms for vaccines, are high. The commercial success of these

See reference 1 for multinational revenue sources; for Japan, served largely by local companies, estimate based on personal correspondence; for GAVI-eligible countries, estimate based on source cited at 3 above; for middle-income countries, authors' estimates.

Chapter 7: Vaccine Economics

products is sparking renewed interest and investment in the vaccine industry, which had appeared moribund in the 1980s. As one example of this, Chiron’s position in the vaccine industry seemed to have been a major factor in Novartis decision to acquire the company in 2005 (6). A second example of growing interest is provided by Pfizer, which had no meaningful presence in the vaccine business until it bought PowderMed Limited, a company focused on DNA vaccine delivery, in 2006 (7). The remainder of the industry’s revenue is attributable to two groups of suppliers: niche players in industrial countries and emerging country suppliers. A small number of primarily domestic suppliers in Europe, North America, and Japan play a minor role in serving small niche markets, for example, for biodefense or travel products. Emerging country suppliers are having a significant impact, serving their domestic markets and other low- and middle-income countries. Eight emerging country suppliers are currently World Health Organization (WHO)prequalified to supply vaccines to United Nations (UN) agencies and the GAVI Alliance with several more actively seeking WHO prequalification (8). Financial measures understate the importance of emerging country firms because they supply large volumes of mature or maturing products at relatively low prices. Their primary customers are low- and middle-income countries or UN agencies procuring on behalf of these countries. These companies are now the largest suppliers of most of the mature expanded program on immunization (EPI) vaccines [diphtheria toxoid/ tetanus toxoid/whole-cell pertussis (DTwP), tetanus toxoid (TT), measles, BCG], and they are playing a critical role in meeting the demands of the global market estimated at around 10 billion doses. UNICEF noted in 2005 that it was buying 64% of its basic EPI vaccines from developing country suppliers and that annual spend with such suppliers had increased almost 250%, rising from $63 million in 2002 to $146 million in 2005. In addition, emerging country suppliers are broadening their production capabilities and product ranges. A number have licensed or are developing conjugate vaccines, specifically Haemophilus influenzae type b vaccines. They have also added quadravalent (DTwP-HepB) and pentavalent (DTwP-HepBHib) combination vaccines to their product ranges, some of which are now prequalified by WHO.

Vaccine Pricing Developing country access to vaccines has been critically dependent on differential pricing (also called tiered pricing, equity pricing, and price discrimination), whereby different prices are charged for the same products in different markets. Under differential pricing, manufacturers charge a much lower price in the poorest developing country markets, allowing these countries access to the product, and charge much higher prices in the industrialized markets, allowing the manufacturer to recoup its research and development expenditures and overheads. Oral polio vaccine (OPV) illustrates the magnitude of the historical tiered pricing. In the 1990s, the highest price at which polio vaccine was offered was, on average, 30 times the lowest price (9)] (Fig. 1). A more recent example is provided by the rotavirus vaccine: Priced at $62.50 per dose in the United States (10), its price to the Pan-American Health Organization (PAHO) is $7.20 per dose (11). This approach to pricing is a common business strategy, practiced in many industries including airlines, which target the business traveler with

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Figure 1 Differential pricing for one vaccine (oral polio vaccine).

higher prices by differentiating the product (e.g., open, changeable tickets, no weekend restrictions) and the sales channels (e.g., purchased through a travel agent). Price tiering in the vaccine market has been facilitated by a number of factors. First, both high- and low-income markets historically purchased the same products, allowing manufacturers to recoup their fixed cost investments through higher prices in high-income countries, while enabling them to charge lower prices, covering only direct costs to low- and middleincome countries. Second, excess production capacity existed for several mature vaccines, enabling manufacturers to serve more marginal markets without requiring any additional investment. Third, as the majority of costs incurred in vaccine manufacture are relatively fixed (i.e., not related to volume), manufacturers have been able to offer widely differing prices once the core fixed costs are covered. Fourth, pooled procurement of large quantities of pediatric vaccines managed through international agencies such as UNICEF and PAHO procuring on behalf of countries provided defined channels that reinforced the pricing. Finally, the acceptance by both suppliers and governments of different prices for different segments of the market has been critical. Tiered pricing has, however, also had some less desirable consequences. In comparison with other products and markets, the UNICEF and PAHO market generated little revenue and even less profit for many manufacturers—two key factors driving a company’s decision to maintain production lines and invest in R&D and new capacity. Companies had difficulty internally justifying their continued involvement in this marginal market. The result was low levels of investment in both vaccine R&D and production capacity to serve the needs of low-income countries. As a consequence, while the tiered pricing model was successful in delivering very low prices for basic pediatric vaccines, it did so at the expense of supply security or rapid access to newer vaccines. The history of tiered pricing provides some warning for the future as the immunization community seeks to accelerate access to the poorest countries. Tiered pricing, and the equity notion underpinning it, can be undermined if the objectives of tiered pricing are too broadly defined, for example,

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striving for early access to a new vaccine at one low price for not just the poorest countries but also all middle-income countries. In the early 1990s, the tiered pricing and vaccine supply model began to evolve, reflecting both changes in the external environment and the stresses induced by the model itself. Probably the most important factor driving this evolution was the growing divergence between products used in high- and low-income countries. As industrial countries transitioned to second-generation products such as acellular pertussis, production capacity began to shift and the overcapacity that had facilitated the supply of high-volume, low-priced vaccine to low-income countries began to disappear. In the 1990s, financing of vaccines for low-income country markets also began changing. The global immunization community formed the GAVI Alliance, and large amounts of funding for the purchase of vaccines for the world’s poorest countries were funneled through this public-private partnership. Much of this funding has been devoted to ensuring that existing but underutilized vaccines against diseases such as hepB, H. influenzae type b, or yellow fever are added to developing country immunization schedules. With promises of new funding to support combination vaccines that both addressed priority diseases and mitigated delivery constraints, developing countries began introducing these new vaccines. Importantly, since these combinations contain wP vaccine, little or no market existed for them in the industrialized world. At the time of introduction in 2000 to 2001, only one supplier, a multinational, made the desired combinations (i.e., DTwP-HepB and DTwP-HepB-Hib), and pricing was at a significant premium both to the sum of the constituent vaccines and the typical vaccine price for this market. By 2007, however, the market had attracted a further three companies, and the price of one of the combinations had fallen by 35% in nominal terms. Finally, the supply base evolved, particularly for the more mature products, which were increasingly produced by emerging country manufacturers. The evolving demand of immunization programs in industrial and developing countries created both the opportunity and the necessity for developing country suppliers to play a much greater supply role than hitherto. In summary, there has been a significant shift in how affordable vaccines are made available to low-income countries. A single historical model of multinational suppliers selling excess capacity at very low prices, with a product offer limited in practice to the most mature products, has fragmented into three models, on the basis of product lifecycle, as follows: l

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Mature product (DTwP, TT, measles, BCG) supply largely provided by developing country suppliers, at low prices. Pricing may be somewhat higher than historically, since the price now must cover the fully loaded costs of production, although developing country manufacturers typically have significantly lower costs per unit than their developed country counterparts. Maturing product supply (DTwP-HepB, DTwP-HepBHib), provided by a mix of industrialized and developing country suppliers. The pricing and the supplier mix will continue to shift over time with emerging firms playing an increasingly important role, as has already been seen with the wP vaccine combinations.

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New or proprietary product supply, likely from industrialized suppliers and requiring significant price tiering to be economically viable. This model is largely notional at this point, since no large-scale implementation yet exists. However, the international public sector is gearing up both financially via AMCs, IFFIm, and traditional donor fundraising (as discussed at the end of this chapter) and operationally via, among other mechanisms, the publicprivate partnerships to introduce new vaccines such as rotavirus or pneumococcal conjugates broadly in lowincome countries. Significant tiered pricing will be critical if availability in the poorest countries is to be achieved, given the higher prices for these vaccines in the industrial world.

BRINGING A VACCINE TO MARKET: THE COSTS, RISKS, AND CONSTRAINTS Each company has a portfolio of R&D projects across which it allocates investments. While the public sector supports a great deal of basic research, private manufacturers have historically financed most of the product development and production scale-up. Assessing and managing the risk associated with product investments is a major part of the pharmaceutical business. The risks span the stages in bringing a vaccine to market and include not only the scientific and technical risks but also political and market-based factors. The combination of known costs and benefits plus possible risks determines a company’s decision to invest in a product or not. Companies continually compare investments across the products in their portfolio. Products with high potential profits and low risk are more attractive than those with limited expected earnings and/or high risk. The expected profits, often measured as a percentage of investment or return on investment (ROI), are a function of expected costs, risks, probable demand, and thus expected profit. Companies must not only ensure each project is viable but that the expected product revenues cover the R&D costs of ongoing and new projects. Given the relatively lower prices available in low-income country markets, manufacturers are, not surprisingly, less willing to accept all the risk associated with vaccine investments for these markets. Understanding the risk-return trade-off, and possible areas over which the public sector has influence, provides the public sector with an opportunity to intervene in a manner that may diminish risks.

Development Costs in a Perfect World There are defined costs that constitute the investment needed to develop and scale up a vaccine and make it commercially available in the absence of any failures, complications, or delays, that is, in a perfect world. These costs include, on the development side, preclinical research, clinical trials, and production scale-up and on the commercialization side, ongoing production, quality control, and marketing. The costs vary on the basis of the particular characteristics of the disease, the vaccine, and the production technology. For example, because of the risks of the relatively rare event of intussusception, regulatory agencies require exceptionally large—and costly—phase III trials for rotavirus vaccine to address this question. The costs associated with product development are incurred in four distinct stages: (i) preclinical research, (ii) identifying a promising vaccine candidate through early

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Figure 2 Baseline investments and decision gates in developing an HIV/AIDS vaccine for one target population. Source: From Ref. 12.

clinical studies in humans, (iii) developing and testing a candidate for a target market, and (iv) scaling up manufacturing capacity for that market. Figure 2 shows the relative magnitude of these investments—turning each new stage into a decision gate initiating a reevaluation of the investment, given the product’s risks and potential return (12). Assuring a vaccine for global use requires two additional stages of investment: (v) relevance—adapting and testing a vaccine to ensure safety and efficacy in additional populations and (vi) supply—ensuring adequate manufacturing capacity and funding/pricing structures to enable broad developing country access. If a new vaccine is to be broadly available at the earliest possible technical and regulatory opportunity, a manufacturer must make development and capital investments explicitly to support supply to the developing world markets. For there to be a business case for such incremental investments, a return must be available either directly from the developing world market or through other special financing interventions. In the case of vaccines for which no significant industrial market exists, such as malaria, it follows that the developing country market and associated financing must fund, and provide a return on, the entire investment.

Vaccine Production Vaccine production has two main stages, bulk production consisting of activities such as growing or producing by fermentation the active ingredients, and purifying the result, and fill/finish consisting of activities such as blending, filling vials, lyophilizing where applicable, labeling, and packaging. All suppliers require production facilities with appropriate quality control and quality assurance that are in compliance with current Good Manufacturing Practice (cGMP), as required by the regulatory authorities. The costs associated with vaccine production can be categorized as variable (unit cost is constant for each vial), semi-variable or batch fixed (costs associated with each batch), and fixed (costs are independent of volume or number of batches and are fixed at the site or company level). Vaccine production is largely a ‘‘fixed cost business,’’ in that the majority of costs are fixed at either the batch or site level (plant, equipment, animals, labor), leaving only a relatively

small percentage truly variable (vials, stoppers, raw materials). In general, the fixed cost nature of vaccine economics means that unit costs fall rapidly with increases in volume. The cost to produce vaccine varies significantly from product to product. The primary factors affecting the cost are outlined in detail below and include the presentation, scale of operations, vaccine inputs, supply location, batch size, and certain vaccine-specific production characteristics. Understanding the economics of production can guide the public sector in its choice of suppliers, its policy decisions such as the optimal presentation of the vaccine, and strategy options such as the timing of commitment to purchase. Timing becomes particularly important because the capacity and batch size for a given vaccine are largely determined at scale-up, and are thereafter expensive and time consuming to increase. Presentation Presentation (e.g., the number of doses per vial, labeling) can be specified by the buyer, within regulatory and equipment constraints, and so must be carefully considered when there are limited resources. As shown in Figure 3, single-dose presentations are significantly more expensive to produce than multidose presentations because of their impact on filling lot size. Lots of single-dose vials require almost the same labor and equipment as multidose vials but with far smaller output. For an industrial country supplier, it has been estimated that singledose presentations added approximately $0.50 per dose to the cost of a liquid vaccine, and $1.00 to the cost of a lyophilized vaccine (13). Scale of Operation A manufacturer’s scale is a function of the total number of doses produced across all products. Operational decisions and, in particular, the number of sites producing vaccines, will also impact the extent to which a given manufacturer’s operations are scale sensitive. The importance of scale is illustrated by comparing suppliers from the United States and Europe. U.S. vaccine suppliers have historically been higher cost than European ones, primarily because of the former’s overall lower volume levels (estimated by the authors at fewer than 100 million doses annually vs. over 1 billion doses for certain European multinationals).

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Figure 3 Average cost per dose across vaccine (multinational producers only) to produce liquid (including TT, DTP, Hep) and lyophilized (including MEA, MMR, Hib) 1 and 10 dose vials. Source: Mercer Management analysis.

Vaccine Batch Size The size of bulk batches is an important cost factor, as shown in Figure 4. Since the cost to manufacture and test a batch is largely fixed, an increase in batch size results in lower per dose costs. Batch size is, however, largely determined at the time of manufacturing scale-up. Once a plant is in place, there are two ways to increase batch size. The first is to add capacity, a process that requires additional capital, may disrupt production and certainly requires regulatory approval and GMP certification. The second is to wait and allow the ‘‘experience effect’’ to drive yield improvements and consequent increases in effective batch size. Although some vaccines, such as recombinant hepB, appear to have experienced very rapid and dramatic improvements in yield, the available data suggest that for most vaccines, it takes years before batches are large enough to serve the developing country market. Given the difficulties of increasing batch size once a plant is built, the optimal strategy to assure

capacity and relative affordability is to influence batch size at the time of scale-up, perhaps through early commitment to purchase. Reliance on Third-Party Components Having to purchase certain expensive components for a vaccine rather than producing them in-house can have a significant impact on costs. As the demand for combination and conjugated vaccines has grown, almost all suppliers have been forced to purchase either antigens or protein carriers from outside firms. The increase in outsourced components significantly impacts the economics of vaccine production. First, outsourcing increases variable cost, so the marginal cost of vaccines with significant outsourced components will likely be higher than those produced entirely in-house. Second, although it is not known, it is likely that these arrangements increase the absolute cost of the vaccine in question, as the overhead costs and profit

Figure 4 Batch size effect on bulk production cost (based on a representative sample of multinational suppliers). Source: Mercer Management analysis.

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margin of the component supplier must also be factored in. For newer vaccines, such as vaccines against human papillomavirus or rotavirus, royalty payments to technology providers may also be significant. These are typically calculated as a percentage of revenues, sometimes escalating as sales milestones are achieved. As a consequence, royalties can significantly increase the variable cost burden, although since they are calculated as a percentage of sales, the impact is lower in absolute terms on products sold at lower prices. Supply Base Location All else being equal, industrial country suppliers are higher cost producers than large emerging suppliers, as wage rates for pharmaceutical labor in lower-income countries are a percentage of those in high-income countries. For example, Panacea, a large Indian supplier, discloses in its financial report that its average salary in 2007 was under $10,000 per person (14). Vaccine Production Characteristics Different vaccines have differing testing and labor requirements, antigen combinations, and production process cycle times, each of which impact the cost of production. However, these differences are less important than overall and batch scale effects. As an example, bulk production of OPV is one of the most expensive manufacturing processes of any vaccine in absolute terms. However, because this vaccine is manufactured by high-scale producers in large batches of multidose containers, it has one of the lowest per dose production costs of any vaccine. Risks In addition to the R&D and production costs incurred, every product faces a range of risks, both foreseeable and completely unpredictable. Should they occur, any one of these risks can necessitate significant additional investment or—if the product fails entirely—result in the total loss of the investment. These risks may increase development, production and sales costs, and ultimately, impact the estimated costs of the product. For example, for a certain vaccine, a planned $100 million investment should result in a cost of $0.50 per dose. However, if there is a one-year delay in launching a clinical trial (adding $10 million), plus a problem with validating the new plant (adding another $20 million), and unexpectedly slow introduction of the product (resulting in both extra inventory costs and lost revenues), a number of unplanned costs must be factored in. Added together, these unexpected events could increase the total costs to roughly $150 million, resulting in a unit cost of $0.80 per dose—significantly over original expectations. Obviously, not all the things that can go wrong, do go wrong. However, cost and pricing estimates must be adjusted to reflect risk. The following are more detailed descriptions of the different types of risks that contribute to the full costs of developing and producing a vaccine: Research and development. Faced with finite human and financial resources to support R&D projects in the pipeline, the decision to invest in one candidate will absorb resources that could have been used for other products. The opportunity cost of supporting one project versus another is significant and must be factored into decisions, with the most significant risk being that no product will be forthcoming, and the program will fail. Given the cost of efficacy trials, companies seek to minimize the risks of a trial failing or being delayed. However, if

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there are not good animal models or correlates of immunity to help predict the probability of the product successfully protecting humans, as, for example, with HIV/AIDS vaccines, then investments in expensive phase III trials for a vaccine candidate are even riskier. In addition, each additional trial to test efficacy in a different epidemiological region will add significantly to the product’s development cost. Moreover, clinical trials conducted in developing countries with different epidemiological conditions may present risks to licensure in industrialized countries that outweigh the potential revenues from the developing country market. Each product may also have unique risks. For example, as the number of antigens in a combination increases, so does the risk that at least one of the antigens will have reduced immunogenicity or efficacy, that antigens will interfere with each other, or that an adverse event associated with one antigen will reduce demand for the combined product. Production scale-up. Companies usually invest in scaling up production capacity before the phase III trials are completed. This early investment is for two reasons. First, it is a condition of product licensure for all biologicals that the company demonstrate that it can make its product in a repeatable (consistent) fashion at commercial scale. Second, to assure timely availability of new products, a production facility must be fully operational as soon as the product proves to be efficacious. However, if the product fails at a late stage, the company risks losing most or all of this investment. Investment in production capacity is particularly difficult because many companies face growing internal competition for these resources. The opportunity cost and risks of building or using a facility for one product versus another must be considered. Sizing a facility appropriately, given uncertainties about future demand, is also difficult and risky. On one hand, a larger facility requires greater investment, but because of economies of scale, it can result in more efficient, lower-cost production in the long run if large volumes of the product are demanded. However, if demand is lower than expected, the facility will be underutilized, the fixed asset investment will be higher than required and unit costs will be higher than would have been the case in a smaller, better utilized facility. On the other hand, a smaller facility requires less total investment but the firm is less able to take advantage of economies of scale and so the long-term cost per dose may be higher. If demand is greater than expected, the company not only loses the market to a competitor but must also invest in resizing its facility, a costly and time-consuming mistake. Regulatory and licensing issues. Not only do vaccine manufacturers face increasingly stringent regulatory requirements but regulatory differences between countries hamper preparation of applications for marketing authorization. Obtaining separate authorizations for each market in which a manufacturer proposes to sell a vaccine is a long, costly, and uncertain process. In addition, the increasing difficulty in finding appropriate regulatory pathways for developing country vaccines produced in the United States and Europe increases the risk to licensure. Most new vaccines are produced in either the United States or European countries. The FDA and EMEA (European Agency for the Evaluation of Medicinal Products) each have a responsibility to regulate products for their home markets. However, these agencies are also called upon to regulate products intended for developing countries that are developed and produced by manufacturers situated within their borders.

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If these agencies decline to, or are prevented from, applying their regulatory expertise for products that will not be marketed within their borders, the risks and costs to the companies will increase significantly. Even once licensed, unexpected adverse events identified through post-marketing surveillance may result in a decline in demand for the product, as in the case of Wyeth’s Rotashield1. The tens of millions of dollars invested in developing and scaling up production for this first-generation vaccine were lost once the product was associated with the risk of intussusception a rare but serious, adverse event. Finally, intellectual property rights (IPR) are intended to safeguard returns on new products, enabling manufacturers to recoup their investment. Weak IPR protection increases the risk (in the eyes of the company that made the initial investment) of multiple suppliers copying and competing for the same market— without having invested in the R&D. This risk may be so significant that it reduces a company’s interest in developing a high-priority vaccine. Concern for IPRs has been less of a problem for vaccines than small molecule drugs, because vaccines are also protected by technical ‘‘know-how,’’ the knowledge of how to scale up and consistently produce a biological product as well as stricter import controls. Commercial production. Once a product is fully developed, licensed, and ready for production, companies face production risks that can occur at any time. Seemingly inconsequential changes to the production materials or process, such as a change in brands of cleaning fluid used to wash out a fermenter, can cause a batch to fail, and hence the investment in the batch to be lost. These failures can be particularly costly, given the relatively lengthy period required to produce and test a batch of vaccine that can be as long as 9 to 15 months. Batch failures and unexpected changes in demand can be costly to manufacturers as they may result in excess inventory, market shortages, or last-minute changes to the production schedule. The fragility of vaccine supply was graphically illustrated when Chiron’s influenza vaccine manufacturing facility

encountered a contamination issue and was consequently unable to supply any product for over a year. Delivery and sales. All investments will be pointless if no market for the product materializes. National introduction depends on whether the immunization delivery systems are able to reach the target population and if there is adequate national and donor funding. A market may ‘‘disappear’’ if there is a change in the perceived risk of a disease or in national immunization priorities. Historically, there has been a great deal of uncertainty about not only which vaccines will be demanded and financed by low-income country governments and partners but also when they will be purchased. The slow and uncertain uptake in low-income countries has greatly increased risk perceived by industry in serving this market. With the advent of the GAVI Alliance providing support to strengthen immunization infrastructure and purchase new vaccines, several countries are still immunizing less than half the target population, and the introduction of new vaccines still depends on national decision makers having the necessary national data in hand. Each Vaccine is Different The relative importance of different risks varies from vaccine to vaccine. Each vaccine faces a unique mix of obstacles that may be best overcome by different types of interventions. Figure 5 shows two key factors that influence how attractive or risky a vaccine is perceived to be—the potential market revenue and the degree of scientific certainty. A vaccine against a disease like malaria, has until very recently, had low scientific certainty (15) and low revenue potential and therefore faces both high development and demand risk. By contrast, a meningococcal A conjugate vaccine intended primarily for Africa has high scientific and technical certainty but a very limited revenue opportunity. In such a case, the primary barriers are the opportunity costs, constraints on capacity, and high demand risk. HIV vaccines, for which the revenue opportunity is high but the scientific and technical certainty is low, represent a challenging

Figure 5 Barriers given degree of scientific certainty and potential market revenue.

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opportunity for commercial vaccine developers. In addition to the very profound fundamental scientific challenges, accelerating its development may require incremental investments to tailor a vaccine to the needs of developing countries once a successful approach is discovered as well as innovative ways to ensure affordable pricing.

INNOVATIVE MECHANISMS TO CHANGE THE ECONOMIC EQUATION Decisions to invest in the development and commercialization of vaccines, while dependent on scientific progress, are based largely on an evaluation of the economic factors: the costs, risks, timing of investments, and expected return on future sales. To influence these economic factors, the public sector is exploring and implementing innovative ways to assess, share, and alter the risks and returns. There is no single solution. Not only do the risks vary by vaccine, but they are also perceived differently by public and private entities. Both the public and private sectors seek to minimize risk. Given the choice, the public sector would avoid all risk, only committing to buy a product once it is developed, widely demanded, and available at a low price. In contrast, the private sector prefers to invest in a product only once solid market commitments were in place, that is, guarantees to purchase the product at an acceptable price. Creative public-private partnerships that share specific risks and costs and protect the interest of all partners are essential if low-income markets are to have rapid access to priority vaccines. There are two generic approaches to accelerating the development and introduction of priority vaccines: push and pull mechanisms. Push mechanisms are those that reduce the risks and costs of investment, while pull mechanisms assure a future return in the event that a product is produced. There has been significant work over the last five years to develop and implement both kinds of mechanisms. Perhaps most significant on the push side is the creation of new product development partnerships (PDPs). PDPs are typically not-for-profit entities mandated to accelerate the development and introduction of a product against a specific disease—such as the Malaria Vaccine Initiative (MVI). PDPs were created recognizing that the absence of a robust commercial market (defined as both need and the ability and willingness to pay for the appropriate product) and the consequent riskiness of commercial R&D investment required creative approaches. PDPs are funded by donors to promote research and development, often through the creation of links between industrial and developing country academic programs, biotechnology companies, and pharmaceutical companies. PDPs have played a particularly important role in facilitating investment in product development such as large-scale clinical trials and process development for diseases of especial and unique importance to the poorest countries in the world (16). Downstream PDPs, such as the GAVI-funded Accelerated Development Introduction Programs (ADIPs) for pneumococcal and rotavirus vaccines, focus more on supply and pricing issues that will affect introduction and uptake decisions. Both upstream and downstream PDPs have filled a critical gap—focusing attention and resources on the neglected development and introduction of priority products for lowincome countries. On the pull side, there have also been a number of partnerships and innovative financing mechanisms developed.

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The GAVI Alliance is, itself, a public-private partnership that was designed to bring together the major partners in immunization; providing them a forum and a means to work together to achieve their common immunization goals. The GAVI Alliance comprises key immunization stakeholders, including developing country and donor governments, civil society, WHO, UNICEF, the World Bank, industry, the Gates Foundation, and others. Through GAVI, partners have channeled large increases in funding, raised through traditional donor contributions and through innovative financing mechanisms. Two interesting new financing mechanisms are the International IFFIm and the AMC. Both are intended to increase the impact of the pull side of the equation.

International Finance Facility for Immunization IFFIm is a way to increase the predictability and quantity of development aid available today. It front-loads donor aid commitments through sales in the bond market, allowing for immediate investment into country immunization programs. Originally proposed by the U.K. Treasury, the International Finance Facility was envisioned as a tool to increase availability of development funding to achieve all the millennium development goals. Immunization was selected to pilot the IFF because it offers a strong rationale for front-loaded funds where returns are large enough to offset the financial costs associated with a bond issuance. IFFIm funding is channeled through the GAVI Alliance to reduce the number of childhood vaccinepreventable deaths in the world’s poorest countries and to scale up delivery systems. By assuring more predictable funding for underutilized vaccines like DTwP combinations, IFFIm funds help attract increased investment by existing and new suppliers to scale up production to serve the low-income country market. Because of economies of scale, rapidly ramped-up production capacity helps reduce costs more quickly than would otherwise occur. IFFIm funds are also provided to governments to strengthen immunization program delivery, thus reducing the risk of disease. The front-loaded IFFIm funds accelerate the scale-up of both vaccine production and delivery systems. GAVI estimates that the current pledges of $3.9 billion of IFFIm funds will prevent 5 million child deaths between 2006 and 2015, and more than 5 million future adult deaths by protecting more than 500 million children in campaigns against measles, tetanus, and yellow fever (17). How Does IFFIm Work? Eight donors (the United Kingdom, France, Italy, Spain, Sweden, Norway, Brazil, and South Africa) have pledged payments totaling $3.9 billion in current dollars over 20 years. The donor commitments are sovereign obligations and are used to guarantee ‘‘immunization bonds,’’ which are sold in the capital markets. The money raised by these bond sales is then channeled through GAVI to support the agreed vaccines and immunization programs in low-income countries. Ultimately, IFFIm will repay its bondholders with the long-term funds committed by the donors. The World Bank is IFFIm’s Treasury Manager providing services such as the development and implementation of financing strategies and funding operations, handling the donor grants and payments, and managing the liquidity of IFFIm funds as they become available. IFFIm had its first bond issuance in November 2006. The $1 billion bond offer was substantially oversubscribed, with

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demand for over $1.75 billion. The second bond issuance occurred in early 2008 in Japan, with others planned as funding is required by GAVI. This innovative financing mechanism has increased both the amount and predictability of funds available today for immunization. This mechanism is helping assure financial support for low-income governments, and tangently, for their vaccine markets.

Case Study: AMC Pilot for Pneumococcal Vaccines The AMC is a mechanism to accelerate the development and availability of priority new vaccines to low-income countries. The design of the AMC builds on a detailed understanding of the vaccine market and the potential risks, costs, and returns to suppliers. It was designed to mitigate the perceived risk to suppliers of investing to serve low-income country market. The following case study of the first pilot AMC for pneumococcal vaccines highlights many of the issues raised in this chapter and illustrates how an understanding of the economics can be used to tailor solutions.

their costs and provide an appropriate return and assurances of reliable demand in the short and long terms.

How Does It Work? The AMC is a tailored commitment by donors to subsidize the purchase of vaccines that meet agreed standards. The total amount available for the AMC and the price per dose are also established at the start of the AMC so that each supplier can assess the benefits of engaging in the AMC against the likelihood of having a product that meets the target profile. The AMC can be divided into the following five basic steps:

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Advanced Market Commitments: Creating a Market The objective of the AMC is to increase the assurance that a vaccine market will exist in low-income countries. The AMC is a financial commitment by donors to subsidize the future purchase, up to an agreed price, of a vaccine not yet available, if an appropriate vaccine is developed, production capacity scaledup and if it is demanded by developing countries. The AMC promotes rather than guarantees a market because countries must still demand the product before any purchase takes place. By guaranteeing in advance that funds will be available to purchase vaccines once they are developed, produced, and demanded, the AMC reduces the risk that governments will not have the resources to pay for a vaccine that is a priority for their national programs. In effect, the AMC underwrites a market that would otherwise be perceived as too small and unpredictable to justify investment by industry. This market assurance creates incentives for firms to invest in priority vaccines for the poorest countries (18,19). Importantly, an AMC is open to all firms that might produce an eligible product. As a result, the AMC does not ‘‘pick a horse’’—selecting one candidate for support, but rather gives equal incentives for any multinational or emerging market firm to accelerate the development and scale up the production to serve the AMC market. AMCs are proving to be attractive to both the public and private sectors because it is a market mechanism that harnesses the resources of private companies. The most critical and difficult issue in designing and pricing the AMC is determining how best to balance the shortand long-term objectives of three stakeholders: developing country governments, donors, and industry. Developing country governments want rapid access to vaccines, but perhaps even more important are predictable prices and reliable vaccine supply in the long term to ensure that national immunization programs continue uninterrupted. Donors want to be assured that their funds are having the maximum health impact by assuring that the AMC fosters increased investments in production capacity and a competitive market able to meet developing country demand at affordable prices, even after the AMC funding is depleted. Finally, suppliers want prices that cover

2.

3. 4. 5.

Donors commit to fund an AMC of a specified size and price per dose for a target vaccine meeting an agreed profile in terms of vaccine effectiveness and public health impact. The target product profile of an eligible vaccine is set by a group of experts called the Independent Assessment Committee (IAC), which draws on expertise in the vaccine community. The target product profile is set to assure the public health value of the vaccine in a low-income country setting. The donors are effectively making a unilateral, legally binding offer that each potential supplier can assess and respond to, depending on their pipeline and interest in the low-income country market. The World Bank will support donors in holding and managing these funds. When a candidate vaccine becomes available, an IAC determines if the vaccine meets the target product profile. The IAC will rely on the WHO prequalification process to inform this assessment. Once an IAC approves a vaccine, the supplier can enter into a pre-agreed AMC supply agreement with GAVI, who will be implementing the operational side of the AMC on behalf of donors. Low-income countries interested in introducing the vaccine into their national immunization program request doses and funding support through GAVI. Countries pay a co-payment for the vaccine through GAVI and donors subsidize the purchase price up to the preagreed AMC price. When the AMC fund is depleted, each supplier is obligated to continue to provide the vaccine at a lower established ‘‘tail’’ price for a specified period or for a specified volume. The tail price can be lowered but not increased in this postAMC period.

AMC Pilot: Case Study of a Pneumococcal Conjugate Vaccine An independent expert group with expertise in epidemiology, public health, and immunization delivery system in developing countries, cost-effectiveness, and industry decision-making processes/vaccine markets (Fig. 6) evaluated six candidate diseases for the first AMC pilot: HIV/AIDS, malaria, tuberculosis, pneumococcus, rotavirus, and human papillomavirus. At the recommendation of this group, a pilot AMC has been designed targeting pneumococcal vaccines (20). The success of the AMC pilot for pneumococcal vaccines will be measured in two ways. One metric will be its ability to influence the decisions of vaccine suppliers to invest in adequate capacity to meet low-income country demand—thus reducing the typical 15-year delay between availability of vaccines in high- and low-income markets. The second metric will be the AMC’s ability to obtain more predictable and

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Minister Heatherwick Ntaba, Minister of Health, Malawi. Prof Adenike Grange, Nigeria, President of International Pediatrics Association. Dr Supamit Chunsuttiwat, Senior Medical Officer, Department of Public Health Thailand, SAGE member. Dr Barakamfitiye, former WHO AFRO Director of Communicable Disease Division and head of Subregional office for West Africa. Ms Merceline Dahl-Regis, Chair of the GAVI Independent Review Committee. Prof Anthony Mbewu, President of the South African Medical Research Council. Dr David Fleming, Epidemiologist, Gates Foundation. Ms Joy Phumaphi, Asstistant Director-General, Family and Community Health, WHO. Dr Paul Henri Lambert, Chair of the Global Advisory Committee on Vaccine Safety. Mr Steve Hurst, Advisor to BioVentures for Global Health. Mr Adrian Towse, Office of Health Economics. Mr Michael Conway, Partner, McKinsey and Company.

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identifies the timing and risks of each investment during the development stage on the basis of the likelihood of success;

estimates the incremental cost of product development, manufacturing, and commercialization for the target market that is not covered by public funding or already committed to serve the high-income country market. analyzes different product profiles and market scenarios, including the likelihood and impact of competition; compares each investment decision with other opportunities and the cost of capital; translates estimates of investment, cost, and return into expected cash flows over time (in net present value terms) and adjusts this cash flow for risk (given the uncertainty of success or failure at each stage of development). However, the financial return on certain life-saving products may be bolstered by intangible value associated with being socially responsible.

The model relies on several critical inputs. As these inputs are only assumptions, the model acts as a tool to keep assumptions organized, transparent, and subject to modification, as better information becomes available. Of particular importance are the estimates of demand, assumptions on each manufacturer’s vaccine development and capacity decisions, broad estimates of cost of goods (COG) sold, and likely competition. l

Figure 6 Expert Group Members.

sustainable supply and prices in the short, medium, and long terms, thus assuring the public health value of this vaccine is realized. In addition, donors have noted that the AMC should promote competition in pneumococcal vaccines by attracting both emerging and multinational manufacturers, encourage innovation, and, of course, assure an efficient use of donor funds. A successful AMC for pneumococcal vaccines depends on setting terms (e.g., size, price, post-AMC supply, and price conditions) that are likely to influence industry’s investments in directions that achieve the AMC’s objectives. To understand how suppliers might value, and therefore react, to the AMC for pneumococcal vaccines, Applied Strategies Consulting developed a model based on valuation methodology commonly used by the industry to compare likely returns (given demand, competition, and other factors) across alternative investments and with the cost of capital (21). Widely vetted by public and private stakeholders in the immunization community, the model estimates returns for suppliers across alternative investments and applying the relevant cost of capital. If the investment under review has a positive return, then it is likely that the company would pursue it, if not, companies would probably choose not to proceed. Thus, setting an AMC price higher or lower, and the overall size of the commitment larger or smaller, should affect the AMC’s ability to attract first, second, or subsequent manufacturers to serve this market. As described by Applied Strategies, the valuation methodology:

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Demand forecasting: Estimates of country demand that predict uptake of a new vaccine, assuming AMC financing, have been developed on the basis of the expected year of introduction and doses demanded, given the population size and expected coverage levels (using coverage of DPT3 as a proxy). It is extremely important to note that although forecasts were developed on the basis of the best available information, there is still a significant risk that actual uptake may be slower or more rapid than forecasted. Strategies that reduce demand risk, such as improved forecasting and more active dialogue with governments about introduction plans, must be implemented in parallel with the AMC. The cost of poor forecasting is high as it results in either too much supply, leaving suppliers with expiring vaccine stocks or underutilized production facilities, or too little supply, leaving countries without vaccine and undermining the credibility of the program. Status of development and capacity: The technology to produce a multivalent conjugated pneumococcal vaccine exists. Investments to develop and produce pneumococcal vaccines have been stimulated to date by large markets in high- and middle-income countries. Sales of the only pneumococcal conjugate vaccine licensed to date, Wyeth’s Prevnar, were $2.4 billion in 2007. A further two companies have candidates in late-stage development, one of which has filed for licensure. The primary goal of these efforts is to supply high-income markets. Hence, the planned capacity will be inadequate to serve global demand, once high-, middle-and low-income countries introduce the product into their national programs. Additional investment in production capacity to serve low-income market will therefore be required in the next four to five years. In addition, for reasons of affordability, at least one emerging country supplier is needed to develop and scale up a pneumococcal vaccine for developing countries in the lifetime of the AMC. To achieve these goals, the AMC is sized to buy vaccine from the first three suppliers to bring an eligible

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vaccine to the market—including a late-entry emerging supplier. Cost of goods: The COGs will determine whether a supplier makes a profit or a loss on each dose sold at the AMC price and in the post-AMC supply and price period. Some of the likely pneumococcal vaccine suppliers will have lower COGs initially. For others, the AMC may motivate process improvements and/or partnership deals to bring COGs in line with AMC prices. Estimates of the likely COGs incurred by each firm now and after 10 years of production are very inexact and must be treated particularly carefully as they are one of the key drivers of any AMC model. Competition: The licensed heptavalent vaccine (Prevnar) has been used in industrial countries to vaccinate more than 30 million children. Capacity is, however, inadequate for global supply and the vaccine is not considered suitable for widespread introduction in developing countries on the basis of the serotypes of Streptococcus pneumoniae circulating in those countries. Two vaccines that extend protection for populations in both developing and industrial countries by adding more serotypes may be licensed by 2009 to 2010. From the 20-plus candidates at different stages of development, other vaccines, including from emerging manufacturers, may come to the market in the following 5 to 10 years.

The AMC must therefore have sufficient funding, and operate for long enough, to purchase vaccine from multiple suppliers, thus providing incentives for these companies to develop, license, and produce the vaccine. Competition is a core objective of the AMC donors as it increases the likelihood of long-term sustainable supply at more affordable prices. Creating an AMC to support competition involves trade-offs, since an AMC with a high price would provide significant returns to the first firm to enter an AMC agreement but would be depleted too quickly for further suppliers to enter the market. A lower price would allow the AMC to operate for longer, providing more time for companies to develop the vaccine, establish capacity, and benefit from the AMC, but potentially delaying earliest availability. The final decisions on the AMC terms will be based on the most robust estimates available. Sensitivity analyses will be run around key assumptions and estimates about the future, for example, slower or faster development of demand, COGs being higher or lower than estimated, and delays in licensing and introduction to the market. If, despite all the efforts to identify appropriate terms the AMC does not provide adequate incentives to obtain the desired investments from industry, the IAC will be responsible for reassessing the AMC and recommending any significant changes to the terms.

Recommended AMC and Post-AMC Terms The recommended size of the AMC is $1.5 billion. The price per dose is to be determined but is estimated to be within the range of $5 to $9 per dose, with developing countries responsible for a small copayment per dose. The first purchases are anticipated to begin in 2009 or 2010 and to last for six to nine years. Once the AMC is depleted, each participating supplier will be contractually obligated to continue to supply the vaccine at a pre-agreed price for a set period. The terms governing the supply and price of pneumococcal vaccines after the AMC is depleted are as important as those in force during the operating life of the AMC. This reflects

the long-term objectives of donors, governments, and industry. The requirements for post-AMC volumes and price are still being determined with the goal of establishing a sustainable supply and demand equilibrium.

Expected Impact of the AMC The broad AMC terms currently under consideration are expected to support purchases from the first three firms to reach the market with an eligible pneumococcal vaccine. The AMC is expected and will be judged by whether the following public health and market goals are achieved (22): Public health goals to help prevent 5.4 million deaths between 2010 and 2030 are as follows: l

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Accelerated introduction of pneumococcal vaccine in lowincome countries beginning in 2010, reducing the historical 15-year delay in the introduction of new vaccines between low- and high-income markets. Sustained supply of affordable pneumococcal vaccines in the long term as measured by adequate supply to meet low-income country demand at prices between the AMC price and estimated marginal costs of production.

Market goals to assure supply at affordable prices to meet demand are as follows: l

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Investments by two or more multinational firms in production capacity to meet the increasing demand from the lowincome countries; One or more emerging vaccine manufacturers to develop, license, and produce an eligible pneumococcal vaccine in the next 10 years; Competition among manufacturers for the developing country market. Investment in new technologies for new and more efficient vaccine production and potentially second-generation technologies (e.g., protein vaccines) tailored to developing country markets.

Next Steps The first AMC pilot is in final development and should be launched in late 2008. Experts and stakeholders from the public and private sector have vetted the concept and the more detailed mechanics proposed for the pilot for pneumococcal vaccines. They have agreed that the AMC can help address the market failures that are inhibiting rapid development, scale-up, and introduction of these vaccines. As this chapter goes to print, six donors, the Governments of Italy, the United Kingdom, Canada, Russia, and Norway and the Gates Foundation have guaranteed $1.5 billion for a pneumococcal AMC. The World Bank and GAVI have agreed to provide fiduciary and operational support, respectively. An IAC has been established and members selected through a competitive process. The target product profile for pneumococcal vaccines has been agreed. Donors are defining the final AMC and post-AMC terms. The agreed terms, processes, roles, and responsibilities will then be codified and launched in a signed AMC framework agreement. Once established, the AMC for pneumococcal vaccines will provide a new tool to help prevent unnecessary pneumococcal deaths in the poorest countries of the world. Importantly, it will also allow a rapid assessment of the value and impact of the AMC mechanism. Donors are already looking to the future

Chapter 7: Vaccine Economics

and have requested the expert committee be reconvened to recommend a vaccine for a second AMC pilot with the goal of testing an AMC’s effectiveness at influencing industry investment decisions in vaccines at earlier stages of development, such as vaccines against malaria or tuberculosis.

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These are but a few of the very real challenges that will require resolute leadership and a deep understanding of the underlying economics to solve. Their resolution is critical to building on the progress of the last decade in making the benefits of modern immunization available to the world’s poorest children.

CONCLUSION The importance of economics in meeting public health goals in the immunization arena is highlighted by recent events that are changing the shape of the vaccine industry. First, corporate interest in vaccines as a field has been revitalized, thanks to the striking commercial success of proprietary vaccines launched in the last several years, with both rapid revenue growth and high profitability. While smaller in absolute terms, new funding for the purchase of existing and underutilized vaccines in the world’s poorest countries has also galvanized the industry, encouraging the development of new products, and attracting new companies. Second, PDPs are bridging gaps in the research, development, and commercialization of priority vaccines for lowincome countries. These partnerships have created significant momentum and are beginning to attract and facilitate broader industrial involvement and build capability in local markets. Third, innovative financing mechanisms are playing an important role in reshaping the market. IFFIm has increased both the quantity and predictability of funds available for immunization programs in low-income countries. AMCs are a market-based mechanism that assure future funding for vaccines for both countries and industry. An understanding of the economic factors encouraging or inhibiting investment in priority products has allowed public agencies to work more effectively with the vaccine industry in both industrial and developing countries to assure developing country needs are met. However, assuring increased investment in R&D of priority products and rapid but sustainable introduction of new vaccines are a challenging task. Innovative mechanisms to better engage not just the larger companies but also small and medium biotechnology and pharmaceutical firms are needed, with a focus on R&D as well as supply. As push and pull mechanisms prove successful and result in more products reaching the market, the public sector will need a more efficient means of supporting governments to prioritize between products. The public sector will also need to find the balance between faster access and higher prices. Higher prices are likely in future, in part at least, because of more expensive production and regulatory processes and because industry will be required to invest in dedicated capacity and perhaps R&D to serve developing country markets. By contrast, industry is likely to be concerned over how quickly the public sector will push to accelerate access through increased number of suppliers and a more rapid transition to the mature product supply model. The greater this concern, the more likely suppliers are to seek to amortize any required investment over a short period of time, pushing up the prices offered. Both the public sector and industry are likely to have concerns about the evolving role of tiered pricing. A key success factor for tiered pricing is likely to be that the lowest prices be available only to the poorest countries. A system that demands one low-tiered price for all low- and middle-income countries, for example, 84% of the world, is not likely to be sustainable for new vaccines.

REFERENCES 1. Company Annual Reports, Merck 2007 Financial Results, January 30, 2008; Wyeth 2007 Annual Review, Wyeth 2007 Annual Report, 2007; GSK investor report for q4 2007, GSK 2007 Annual Report, 2007; Sanofi-Aventis Form 20F SanofiAventis Form 20, December 31, 2007; and Novartis Novartis 2007 Annual Report, 2007; 2000 figure and 1990s growth rate from Lessons Learned New Procurement Strategies for Vaccines, June 28, 2002. 2. Siwolop S. Big Steps for Vaccine Industry., July 25, 2001. Available at: http://query.nytimes.com/gst/fullpage.html?res=9C03E6D71 F3AF936A15754C0A9679C8B63&sec=&spon=&pagewanted=all. 3. Wyeth Annual Report, 2007. Available at: http://library.corporate-ir. net/library/78/781/78193/items/283759/Wyeth_AR_ 07_lo.pdf. 4. Global Alliance for Vaccines and Immunization (GAVI) Alliance website Global Results, August 2007. Available at: http://www. gavialliance.org/performance/global_results/index.php. 5. For companies exiting the vaccine industry, see, for example, Big Steps for Vaccine Industry; Fresh Approaches And Technology Ignite New Interest, April 2008 and Big Steps for Vaccine Industry; Fresh Approaches And Technology Ignite New Interest, Summer 2002; Also see Mercer Management Consulting, ‘‘Lessons Learned: New Procurement Strategies for Vaccines. Final Report to the GAVI Board,’’ June 6, 2002. 6. Greil A., Novartis to Buy Rest of Chiron In Deal Valued at $5.1 Billion. October 31, 2005. Available at: http://www.mindfully. org/Industry/2005/Novartis-Chiron-$5_1B31oct05.htm. 7. Press Release Pfizer Expands Commitment to Fight Infectious Diseases. October 9, 2006. Available at: http://www.powdermed. com/pdf/powdermed-pfizer%20acquisition%20release%20final% 20091006.pdf. 8. GAVI Alliance. Developing Country Vaccine Industry, 2007. Available at: http://www.gavialliance.org/about/in_partnership/ developing/index.php. 9. Mercer Management Consulting. Presentation on the ‘‘Economic Framework for Global Vaccine Supply: Optimal Methods to Meet Global Demand.’’ Presented at the CVI/Rockefeller Conference on Global Supply of New Vaccines, Bellagio, Italy, February 3–7, 1997. 10. Widdowson MA, Meltzer MI, Zhang X, et al. Cost-effectiveness and potential impact of rotavirus vaccination in the United States. Pediatrics 2007; 119(4):684–697. 11. Andrus J. Introduction of New Vaccines: Perspectives from PAHO’s Pro-Vac Experience. Presentation, December 2007. Available at: http://www.havmeeting.info/pdf/HAVMIAS84JonKimAndrus. pdf. 12. Batson A, Ainsworth M. Obstacles and Solutions: Understanding Private Investment in HIV/AIDS Vaccine Development. Bull World Health Organ (Geneva) 2001; 79(8):721–728. 13. Mercer Management Consulting, ‘‘Lessons Learned: New Procurement Strategies for Vaccines. Final Report to the GAVI Board’’ June 6, 2002. 14. Panacea ‘‘Revised Guidelines and Forms for Countries to Use in Submitting Proposals to GAVI and the Vaccine Fund.’’ August 2001. 15. Malaria Vaccine Initiative, ‘‘Vaccine Challenges: Why is Developing a Malaria Vaccine Difficult?’’, January 25, 2002. Available at: http://www.malariavaccine.org/mal-vac2-challenge.htm. 16. Hughes B. Vaccine partnerships to tackle neglected diseases. Nat Rev Drug Discov 2008; 7:277–278. Available at: http://origin.www. nature.com/nrd/journal/v7/n4/full/nrd2564.html.

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17. IFFIm. ‘‘Why IFFIm?’’. Available at: http://www.iff-immunisation. org/. 18. Tremonti G. Advance Market Commitments for New Vaccines: A Tool in the Fight Against Disease and Poverty. Report to the G8 Finance Ministers, London, U.K. Advanced Market Commitments for Vaccines; 2005. 19. Levine R, et al. Report of the Center for Global Development Advance Market Commitment Working Group, ‘‘Making Markets for Vaccines: Ideas to Actions,’’ 2005. 20. World Bank and GAVI, ‘‘AMC Pilot Proposal,’’ prepared for the First Donor Working Group meeting, Rome, Italy. Pilot AMC for Pneumococcal Vaccines, September 7, 2006. Available at: http:// www.vaccineamc.org/files/AMCPilotProposal.pdf.

21. Applied Strategies Consulting, Financial Implications and Risk Model: Overview and Analysis Results, Presentation to the Second Donors Meeting on AMCs, London, UK, November 2006. Available at: http://web.worldbank.org/WBSITE/EXTERNAL/ TOPICS/EXTHEALTHNUTRITIONANDPOPULATION/ EXTVACCINES/0,,contentMDK:21132122~pagePK:210058~ piPK:210062~theSitePK:384076,00.html. 22. World Bank and GAVI, ‘‘Framework Document: Pilot AMC for Pneumococcal Vaccines,’’ background for Second Donor Working Group meeting, London, UK Pilot AMC for Pneumococcal Vaccines, November 9, 2006. Available at: http://www.vaccineamc. org/files/Framework%20Pneumo%20AMC%20Pilot.pdf.

8 Development and Supply of Vaccines: An Industry Perspective Michel Gre´co Independent Vaccine Expert, Formerly Aventis Pasteur, Lyon, France

Luc Hessel Policy Affairs, Europe, Sanofi Pasteur MSD, Lyon, France

INTRODUCTION The inception of the 21st century represents a period of great change in the geopolitical approach to vaccinology. Empiricism has given way to more scientific strategies using the most innovative technologies, and several new vaccines against diseases that were not previously preventable are now available. This leads us to reexamine some of the priorities that until now have driven the conception, proposition, and adoption of vaccination policies. The introduction of new vaccines has and will have a major impact on national budgets and the ways in which vaccination programs are funded. These changes in the socioeconomic environment of vaccination encourage undertaking a fundamental overhaul of prevention policies and their financing. However, this in turn will affect the structure and dynamics of the vaccine market and the challenges faced by the vaccine industry. The future is bright for vaccinology, on the basis of a series of strengths and opportunities. However, there also exist weaknesses and threats, and they must be addressed. The major strength lies in the current and future major scientific advances relevant to vaccines. Indeed, vaccines currently represent the most innovative part of the pharmaceutical industry. Opportunities reside in improving the public’s perception of the value of vaccines and the necessity for their development and implementation, triggered by concerns over existing infectious disease threats, emerging infectious diseases, bioterrorism, pandemic influenza, and other factors. From an industrial perspective, a weakness derives from the relatively minor contribution that vaccines make overall to the pharmaceutical market, where they are often viewed as overly expensive and as an expenditure rather than a cost-effective investment in public health. There is also the imbalance between the pressing needs of developing countries and considerations of cost-effectiveness in developed countries. As for uncertainties that concern the vaccine industry, they include shortfalls in funding for programmatic national vaccination policies and the negative safety perceptions held by segments of the general public in industrialized countries. Some other uncertainties relate to production capacity issues and research challenges.

THE WORLD VACCINE MARKET, PRESENT AND FUTURE The notion that vaccines constitute a credible and reliable financial market is fairly recent. Apart from smallpox (Jenner), rabies (Pasteur), and inactivated whole-cell parenteral typhoid vaccine (Wright and Pfeiffer) (see chap. 1), almost all vaccines were discovered in the 20th century, mostly in the second half of the century. During the first years of the 21st century, six new vaccines have already been licensed, expanding the number of vaccine-preventable diseases from 22 to 25. Within the pharmaceutical industry, vaccines were historically set apart because of the close interactions between industry and public health authorities in relation to these products; for many years, they were basically viewed as not-for-profit or minimal-profit products (1). As a result, as recently as the late 1980s, most pharmaceutical companies were leaving the field of vaccines (2). A number of factors have combined to reverse the tide so that today vaccines are a much more competitive commercial area (3). Nevertheless, the field remains at best a ‘‘qualified’’ market that is strongly regulated and has high entry barriers and supply constraints, is largely ‘‘monopsonistic’’ in nature, and must adjust to specific price-setting mechanisms (4). The vaccine ‘‘market’’ actually comprises three major subsets: The so-called ‘‘closed markets’’ where local producers supply the local needs with mostly Expanded Program on Immunization (EPI) monovalent vaccines, which generally cannot be exported, mainly for regulatory reasons, and the value of which cannot be readily assessed (5,6). This segment is evolving with the emergence of new major economies such as China, Brazil, and India, and the development of strong local producers. In the medium term, one should expect increased competition from and in these markets. ‘‘Donors’ markets’’ managed by international organizations [WHO, UNICEF, Pan American Health Organization (PAHO), Global Alliance for Vaccines (GAVI, now called the GAVI Alliance), etc.] who buy mostly EPI

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Figure 1 Market share of vaccines in the global pharmaceutical market in 2008 (estimated).

vaccines for routine or mass immunization in developing countries, generally in multidose presentations and at tiered prices (7–9). Even though the commercial value of this segment has increased, it remains a minor part of the overall vaccine market in financial value, if not in the number of doses. This situation should change dramatically in the coming years with the implementation of the WHO Global Immunization Vision and Strategies (GIVS). The goal of GIVS is to save 10 million lives, thanks to vaccination between 2006 and 2015; this would represent a two-thirds reduction of mortality in children under five years of age at the cost of E800 million. ‘‘Commercial markets’’ are markets—public or private— strongly regulated where competition does exist and where pricing is implemented in a more classical way so that companies can fund their research and develop-

ment (R&D) activities, invest in state-of-the-art industrial facilities, and return a profit to the companies’ shareholders. This is by far the largest part of the global vaccine market in monetary value (1,3,4,10). It is this segment of the global vaccine market that will be analyzed in more detail in this chapter. The commercial market is a fast-growing, concentrated market. As recently as the late 1980s, the commercial market was worth less than $1 billion worldwide. At that time, it was mostly spread among basic bacterial vaccines and some viral vaccines, among many small national public institutes or companies and vaccines were generally sold at low prices. In the course of the last 20 years, this market has increased 20-fold to exceed US$14 billion in 2008 (estimated). But even at this level, it must be emphasized that vaccines represent only slightly more than 2% of the global pharmaceutical market (Fig. 1), around the same figure as the single lead pharmaceutical product. This market is currently shared among a few vaccine categories, the first four of which represent two-thirds of the global vaccine market (Fig. 2). Linked to the generally higher prices for vaccines in North America, this part of the world represents over 40% of the market value, with Western Europe and the ‘‘Rest of the World’’ representing 30% each (Fig. 3). Although the value of the Japanese market is significant, the number and type of vaccines used there is notably more limited compared with most other developed markets. It appears likely that this situation will change substantially in the coming years. Looking to the future, it is expected that the market will continue to grow at double-digit rates throughout the decade, possibly to reach $25 billion by 2015 (Fig. 4) (1). In this chapter, some of the main reasons for the growth of the vaccine market and industry, past and future, will be analyzed (1,11).

Innovation Innovation remains the key driver of growth. What drove growth in the last 25 years was primarily the introduction of

Figure 2 The top four vaccine families constituted two-thirds of the market in 2008 (estimated).

Chapter 8: Development and Supply of Vaccines: An Industry Perspective

2.

Figure 3 Geographical breakdown of the global vaccine market 2008 (estimated).

3.

4.

5. Figure 4 Evolution of the global vaccine market from a ‘‘nonmarket’’ to a substantial one.

major new vaccines, such as recombinant Hepatitis B, polysaccharide-protein conjugate vaccines against Haemophilus influenzae type b, pneumococcus, and meningococcus, Hepatitis A, attenuated varicella and zoster vaccines, live rotavirus vaccines, and human papillomavirus (HPV) virus-like particle vaccines. Another contributor to the growth of the market has been the array of innovative combination vaccines for pediatric and adult use, both for primary immunization and for booster use (12). The future should witness an acceleration of innovation despite increasing risks. Indeed, currently there are approximately 200 new vaccine projects under study globally, the vast majority of which are covered within the chapters of this book. Obviously, many will not make it to the market (13–15) and some will not survive as products after licensure and introduction (e.g., the fate of Rotashield1 rotavirus vaccine) (16). Nevertheless, never before has there been so much research activity in this field. Some of the reasons responsible for stimulating this research include the following:

1.

Vaccines remain primarily directed to the prevention of infectious diseases, including bacterial infections that antibiotics are supposed to cure. The widespread use of antibiotics has led to increasing resistance of microorganisms

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to these compounds. Thus, in some instances, the overuse of antibiotics ‘‘opens the way’’ for vaccines. This was true in the development of conjugated vaccines against H. influenzae type b and is even truer for pneumococcal vaccine (17). This is likely to extend to other bacteria like group A Streptococcus pyogenes, Staphylococcus aureus, and some others for which vaccines would provide both appropriate medical and economic answers. The progress of medical science and an improved understanding of the immune system have enhanced knowledge of the etiology and pathogenesis of infectious diseases and on how the body defends against infectious agents. It has been shown that a number of chronic diseases have an infectious origin, such as duodenal ulcers (Helicobacter pylori) and perhaps coronary heart disease (Chlamydia pneumoniae), and a number of cancers (e.g., HPV in cervical cancer, Epstein Barr virus in nasopharyngeal carcinoma, and Hepatitis B virus in hepatocellular carcinoma). The introduction of primary prophylaxis of cervical cancer less than 20 years after having identified the cause of this cancer is a major achievement. Whereas heretofore vaccines have been used mostly as preventive agents, they are now being explored as possible therapeutic agents, consequent to scientific developments and to economic burden considerations (18). The emergence of biotechnology and molecular biology has led to the development of new tools allowing the more rational design of new products. To cite one example, the development of ‘‘reverse vaccinology’’ allows an accelerated selection of potential protective antigens (19,20). The sequencing of the human genome further broadens the field of development and use of potential new vaccines. Other aspects such as the development of new immunization systems also contribute to this innovation process: combination vaccines, safe syringes, needle-less injections, novel adjuvants, transcutaneous administration, mucosal delivery, transgenic animals and plants, DNA immunization, and many more developments will, over time, drastically change the approaches to immunization.

These and other reasons explain why innovation will remain a key driver in expansion of the vaccine industry, generating many new projects and, ultimately, many new licensed products (Fig. 5).

Demography Demography is another key growth driver for the vaccine field in multiple ways. Volume-wise, immunization is primarily driven by the continuing growth of the population in less developed countries. More than 80% of the vaccine doses produced in the world are currently directed to these markets. In industrialized countries, apart from innovation, the growth of immunization is largely driven by the ‘‘senior citizen’’ segment of the population that is continually expanding. Already, with vaccines directed at the prevention of influenza, pneumococcal infections, and zoster, in addition to the expanding use of booster immunization, this segment has a large growth potential. In a more general trend, immunization, which used to be directed mostly to infants, is extending to all stages of life, including adolescents and young adults (21), as well as the elderly. Travel and migrations constitute another major potential source of clinical infectious diseases, which are quite often

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Equity

Figure 5 Evolution of the vaccine market structure: past and future.

In a global world, it is becoming increasingly unacceptable to see children in poor countries continue to die from vaccinepreventable diseases when the needed vaccines exist and can often be obtained at reduced prices. Mobilization of the international community, including the World Bank, WHO, UNICEF, and a number of public and private organizations such as the Gates Foundation, and industry, in the late 1990s culminated in the formation of GAVI (23–25). This alliance of partners is demonstrating that the whole world can benefit from the tremendous progress of vaccinology (13,26). Industry is convinced that this initiative has a good chance of achieving sustained success because all the actors are present; a methodology exists to plan and implement activities and to guarantee that the results are monitored. Importantly, funding has been available from the onset to strengthen immunization services and to finance the introduction of new vaccines for the world’s least developed countries through financing provided by the Vaccine Fund (25,27). In chapter 7, additional details are provided on the financing mechanisms that have been developed since 2000 to achieve the goal of universal immunization globally.

Bioterrorism vaccine preventable. This includes specific ‘‘at-risk’’ populations such as deployed military contingents. The overall consequence of these demographic trends is an ever-expanding demand for existing and new vaccines and a market structure where the balance of sales is progressively turning to adult/senior immunization, as opposed to infants’ vaccination (Fig. 6).

Prevention Among other key factors impacting the demand for vaccines is the realization that prevention makes sense both from a medical standpoint (avoiding disease, resistance to therapy, side effects of drugs, etc.) and from a societal and economic standpoint (pay a little now to reduce the public health bill down the road both in terms of direct and indirect costs). In this respect, a better recognition of the ‘‘value’’ of vaccines both in the developing and developed worlds will contribute to expanded vaccination coverage and to increased research efforts for new vaccines (22). Greater use of pharmacoeconomic studies should also increasingly justify the expansion of immunization.

The emergence of bioterrorism in the late 1990s and the cataclysmic events of ‘‘September 11, 2001’’ stimulated demand for vaccines against bacteria or viruses that can be used as agents in civilian bioterror, including anthrax (28,29). This has led many authorities to question whether immunization could ever be stopped for diseases such as polio where final eradication is in sight, not to mention more exotic diseases.

Health Threats In a globalized world, where pandemics can spread very fast (migrations, transports, viral mutations, etc.), diseases such as severe acute respiratory syndrome (SARS) and avian influenza have triggered new initiatives by health authorities. For influenza, whether nationally or internationally, these initiatives have led to a major increase in the market size for these vaccines and in considerable amounts of funding for research (30). In concert, all the above-mentioned factors indicate that the demand for existing and new vaccines will remain strong in the foreseeable future.

THE VACCINE INDUSTRY

Figure 6 Global vaccine market age segmentation in 2008 (estimated). In 2015, adolescent and adult vaccines are expected to constitute 60% of market value.

The vaccine industry is fairly young. It finds its origin in national public institutes or companies that were set up to ensure that the basic health needs would be provided to the population, mostly for the prevention of diphtheria, tetanus, pertussis, and poliomyelitis. With the development of additional new bacterial and viral vaccines, recombinant vaccines and, more recently, new technologies, and with the need to scale up production and invest heavily in capacity and compliance, a number of local producers—whether in developed or in less developed countries—have disappeared or been acquired. As a consequence, the vaccine industry has become increasingly concentrated among a few key players (1,9,10,31) (Fig. 7). Sanofi Pasteur is probably the oldest one among the major actors, with the broadest vaccine range and geographical coverage. It results from the merger in the late 1980s of Institut Me´ rieux, Pasteur Vaccins, and Connaught Laboratories

Chapter 8: Development and Supply of Vaccines: An Industry Perspective

Figure 7 Manufacturers’ estimated market shares 2008. (The sales of Sanofi Pasteur, a European joint venture between Merck and Sanofi Pasteur are split 50/50 between the two companies.)

Limited. Initially, a subsidiary of the Rhoˆne-Poulenc Group, Pasteur Me´rieux Connaught, changed its name to Aventis Pasteur (following the merger of Rhoˆne-Poulenc and Hoechst) and more recently to Sanofi Pasteur following the acquisition of Aventis by Sanofi (2003). GlaxoSmithKline (GSK) Biologicals originated in a small Belgian operation called RIT, which was acquired by SmithKline and French and, subsequently, through mergers, became SmithKline Beecham (SB) Biologicals and GSK Biologicals. A small operation until the mid-1980s, the company grew extensively on the basis of its Hepatitis vaccine franchise (recombinant Hepatitis B and Hepatitis A). It then broadened its product range to include infant combination vaccines, expanded international distribution, and development of a broad range of new vaccines, including varicella, rotavirus, HPV, pneumococcal and meningococcal conjugate, and candidate vaccines against malaria and tuberculosis. It is also becoming a major player in influenza vaccine with the extension of its current production facilities in Germany, investments in the United States, and the acquisition of ID Biomedical in Canada, as well as R&D investments in cell culture and adjuvant technologies that have already borne fruit. Merck Vaccines is a specialized division of Merck & Co. that has long been the U.S. market leader. It expanded operations in Europe through a joint venture with Sanofi Pasteur MSD and is entering the international markets with a series of new vaccines against varicella, zoster, rotavirus, and HPV. These 3 players represent each between 20 and 25% of the world commercial vaccine market. A fourth company, Wyeth Vaccines, long a market leader in the United States, actually stopped distributing some basic pediatric vaccines before staging a resurgence at the end of the last century with licensure of a seven-valent pneumococcal conjugate vaccine (Prevnar1), as well as a meningococcal C conjugate vaccine (in Europe). From a market perspective, Prevnar was the first ‘‘blockbuster vaccine.’’ The fifth company, Novartis Vaccines, resulted from successive mergers between national companies in Italy (Sclavo), Germany (Behring), and the United Kingdom

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(Evans-Medeva/PowderJect) within Chiron vaccines in the United States. Chiron vaccines was in turn acquired in 2007 by Novartis, which is strengthening its position in influenza and polysaccharide conjugate vaccines. Other less prominent actors have resulted from the concentration of several smaller companies, such as Baxter Vaccines (Immuno, Nava, etc.), Crucell acquiring Berna Biotech and SBL. The remaining smaller manufacturers in developed countries are mostly dependent on major manufacturers for supplying their needs and are generally meaningful only in their home markets (Australia, Netherlands, etc.). One can expect the future to bring even more amalgamation of vaccine manufacturers, the emergence of new players, and the likelihood that the relative positions may well change again linked to the pace of innovation in the various companies and the success of the development projects that they have chosen to support. This is clearly illustrated by the recent involvement of Novartis, AstraZeneca (which acquired MedImmune), and Pfizer (acquiring Powdermed) in the vaccine business. Yet other new actors are likely to appear. Some pharmaceutical companies may become involved in a selective manner, limiting their focus to a few highly innovative, high revenue– generating products. Biotech companies are increasingly becoming involved in the innovation process as major companies outsource an increasing share of their R&D budgets. Some new vaccine companies (e.g., Intercell in Austria) will mature and begin to manufacture multiple products, and some will try to progress to fully integrated operations, but this will remain a major challenge. Also, producers from major less developed countries (such as Serum Institute of India and other Indian producers, Biopharma in Indonesia, Chinese producers, Brazilian companies, etc.) will become more prominent, especially as developed-country manufacturers rationalize their product ranges, particularly with respect to older traditional vaccines such as measles, Bacillus Calmette-Gue´rin (BCG), and diphtheria, tetanus, and pertussis (DTP) (32,33). Over time, the developing-country manufacturers will increasingly gain expertise and become more competitive with the main multinational actors. This is already the case for traditional, low-priced vaccines, for which the Developing Countries Vaccines Manufacturers Network (DCVMN) has become the largest supplier of UNICEF with respect to the number of doses needed for many monovalent and some multivalent vaccines. For more innovative and costly new vaccines, it will take more time. The above will also lead to increased cooperation either on specific programs or more globally between developing- and industrialized-country manufacturers and to a broader globalization of the vaccine market.

DEVELOPMENT AND SUPPLY OF VACCINES: MAJOR ISSUES FACING INDUSTRY A large demand—present and future—and a limited number of existing or potential suppliers combine to pose a challenge for meeting and facing a complex process of researching, developing, manufacturing, and supplying the vaccines needed for the world at large. Without elaborating on each step of the process, some of the key issues or hurdles for industry will be discussed below.

The Research and Development Process Although the different stages of the R&D process (Fig. 8) may look quite similar for vaccines, as for pharmaceuticals, they are

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Figure 8 General concept of development phases.

very different in a number of ways and have substantially changed over the past few years. Lead Generation The likelihood of completing and bringing a vaccine development project to licensure used to be fairly predictable, with a chance of success ranging from 10% to 100%. In contrast, for the pharmaceutical industry, the likelihood of analogous success with a candidate drug was only ca. 0.0001%. The ‘‘easy’’ vaccines have already been developed. Nevertheless, the advent of biotechnology has resulted in more than 200 new vaccine clinical development projects worldwide and many more in the preclinical stage (1,11). By and large, the new vaccines under development are more rationally designed and avoid some pitfalls of earlier traditional vaccines. Yet, in practice, one finds a much higher failure rate and an increasing difficulty in expeditiously determining correlates of efficacy, thereby leading to a delay in obtaining a proof of concept (Fig. 8) (2,13–15,31). In many cases, the proof of concept is only reached at the end of large efficacy trials, thereby leading to excessive R&D costs. Increasingly, major vaccine companies are sourcing their candidate vaccines from the academic and biotechnology world to the point where the role of researchers in these companies is becoming one of testing and evaluating external programs and projects more than generating new projects in-house.

Clinical Development In the clinical field, timelines and costs have escalated substantially consequent to more stringent regulatory, safety, and quality requirements. Especially, the need to detect very rare but clinically significant side effects for vaccines has led to huge increases in the number of subjects who must participate in clinical trials (e.g., the size of rotavirus vaccine trials) (34,35). Where a few hundred cases were needed a decade ago, the numbers have climbed to the thousands to sometimes reach 20,000 in the mid- to late 1990s. More recently, following the withdrawal of Wyeth’s rotavirus vaccine, two new candidate rotavirus vaccines had to include *65,000 subjects in their phase III trials (34,35). With respect to timing, 10 years is now considered to be a minimum for completion of the clinical development of a new vaccine (Fig. 9), assuming a good initial

Figure 9 Research-to-Launch is a lengthy process, typically spanning 8 to 12 years.

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Figure 10 Vaccine development costs: the typical profile for a $500 million project.

understanding of the immune mechanism for protection against the disease. It is much longer when the pathogen, its pathogenesis, and the relevant immune responses are not well understood (e.g., HIV, malaria, tuberculosis, etc.). Where the development costs of vaccines used to be quite cheap compared with pharmaceuticals, the costs now run in the hundreds of millions of dollars and clinical trials sometimes can only be conducted with the support of large public sector institutions, especially for the more costly late development phase (10). The example in Figure 10 illustrates a typical distribution of costs among the various phases. These costs are based on a project of average size and a fairly straightforward process with no major difficulties. Not included are the likely increased amounts required to be spent on the regulatory process and on post-marketing surveillance (PMS). For these reasons, careful planning is needed to allow as many activities as possible to be run in parallel. Such management is a key to the competitiveness of companies in terms of time to market (Fig. 11).

Formulation Development To comply with increasing safety concerns and higher quality standards, new vaccines have to avoid inclusion of a number of agents that have long been shown to be useful and sometimes indispensable for vaccine formulation (36). Bovine material (mostly fetal calf serum) has been used in most vaccines as a growth medium. Following the bovine spongiform encephalopathy (BSE) scare, this material had to be sourced from so-called ‘‘BSE-free countries.’’ For new vaccines, avoiding bovine material has become the rule. Similarly, human albumin has long been used in the formulation of a number of vaccines to improve their stability. For similar reasons, the trend is to replace it by recombinant substances or other materials deemed more acceptable. It has long been known that to assure the immunogenicity of certain vaccines, adjuvants were needed and aluminum salts (hydroxide or phosphate) provided acceptable results. Now, questions are being raised about the appropriateness of using these adjuvants even though little or no proof has been provided to show that alum has noxious properties. Many

Figure 11 The Research-to-Launch process involves many functions.

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companies are involved in the development of new adjuvants. In most cases, these candidates yield encouraging, positive results in animals. However, moving from animal models to humans is problematic, as has historically been the case with promising new adjuvants. Nevertheless, this is clearly an area where new developments are under way, and in the coming years, we expect to witness the licensure of new adjuvants even though the process will remain slow because of the need to assure the proper balance between immune enhancement properties and safety concerns. Preservatives have been used for many years to protect vaccines from bacterial contamination during the manufacturing process or downstream during product handling. This has proved necessary especially for multidose vaccines or to support WHO’s ‘‘open vial’’ policy. In the year 2000, the American Academy of Pediatrics ruled that the use of mercury salts in vaccines was to be avoided (37) while not establishing a causal relationship between the presence of these compounds and specific undesirable or toxic effects in human. In developed countries, this has not only led to an overall move to singledose vials that contain no preservatives but also resulted in a strengthening of manufacturing procedures. Even though feasible, it has led to major disruptions in the supply of vaccines to these markets and to substantial price increases. Should these decisions be fully enforced across the world, this would imply major shortfalls in the supply of vaccines to less developed countries, with the disappearance of a number of multidose vaccines, a resulting shortage in manufacturing capacity for monodose vaccines, and a steep increase in prices. In the manufacturing of vaccines, companies also tend to rely (for both security and regulatory reasons) on proven technologies despite the fact that some of these may be viewed as outdated. This can be the case for egg-based vaccine production (as for influenza vaccines), where public authorities would prefer the use of more predictable and consistent substrates such as cell culture. Replacing reliable manufacturing processes with more modern techniques is a painstaking, stepwise process in which there is often a tension between scientific and economic objectives. Vaccine delivery systems are another area where one would expect rapid progress to move away from needles because of fear of injections with needles, contamination risks, hazards in the disposal of used, contaminated sharps, etc.). In practice, despite extensive research in various delivery systems (microneedles, oral route, nasal route, transcutaneous devices, jet injectors, aerosols, etc.), syringes still remain the standard (consistency, compliance, etc.). Among other sensitive points, contrary to the general pharmaceutical industry trend, the vaccine industry still has to make extensive use of animals for its toxicity studies and for quality control of the potency of vaccines (38). While it has been working hard to develop alternative cell culture–based solutions, industry still has to use mice, rabbits, and monkeys. Linked to pressures from lobbying groups, this is becoming increasingly sensitive and difficult and could also negatively impact the supply of vaccines. In response to pressures from regulatory authorities, in particular the FDA, companies have had to reinforce the quality and strictness of compliance of their processes (39). This has required substantial financial and human investments to address the stricter requirements, including more rigorous documentation. Some of these changes add substantial costs and time to the production of vaccines.

The Manufacturing Process Many issues relevant to vaccine R&D are equally applicable to ultimate large-scale manufacturing, with the analogous progression of scale-up from the bench level to the pilot stage and finally to the industrial stage, all the while ensuring consistency (2,10,15,38–42). This raises scientific and technical issues, including validation, testing, and quality assurance. It also raises capacity and financing issues linked to heavy investments in buildings, facilities, equipment, and head count. In this respect, vaccines are clearly much more labor intensive than pharmaceuticals. It is quite common to have 50% or more of the total head count of vaccine companies residing within the industrial area. Also, apart from direct production, many more competencies are needed. The rule of thumb is that for one person employed in production, at least one is needed in quality control. Quality and compliance are enforced both internally [by ever more stringent procedures of good manufacturing practices (GMP)] and externally [through inspections by regulatory authorities that can result in ‘‘remarks,’’ ‘‘warnings,’’ ‘‘consent decrees,’’ or even outright closure of manufacturing plants (39)]. This has now affected all major manufacturers in one way or another and is adding to the issues relevant to costs, supply, and productivity. In vaccine manufacturing, time is crucial in both the short and long terms. This highlights the necessity for good forecasting and good planning while always keeping in mind the uncertainties linked to biological production. In the short term, moving from bulk production to the distribution of the vaccine as a finished product is not a simple operation; it takes anywhere from 9 to 22 months (with the exception of influenza vaccine) to manufacture and release a vaccine (Fig. 12). Up to 70% of this production cycle is dedicated to quality control testing. In practical terms, this means that an inaccurate forecast, a poor production planning, a batch failure, a change in recommendations by public health authorities, delays in batch releases, etc., will almost automatically translate into delays of months or years at market level. In the longer term, investing in new manufacturing equipment or facilities is also not a simple exercise and can take anywhere between two years for a new packaging line (studying, ordering, installing, validating) and five years for a new facility and seven years for a new manufacturing site, assuming no major complications (Fig. 13). In practical terms for the vaccine manufacturer, this means having to take a major financial risk without knowing whether the development project (e.g., results of a phase III vaccine efficacy trial) will be successful and will yield an actual product. These time and cost elements have become even more critical in recent years since manufacturing capacities have often been saturated. Therefore, new investments have to be assessed on the basis of dedicated capacities, using the lowest market prices to establish the payback and the net present value of the investments. The relevant resulting product costs then have to be computed on a ‘‘full-cost’’ basis, not just on marginal costs as could be the case when capacities were not saturated.

The Supply Process Having performed research on developed and manufactured vaccines, industry must supply them to the markets. Indeed, there is no such thing as a unified vaccine market, but rather several markets, each with its own specific features, as already mentioned: poor versus developed countries; public versus

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Figure 12 Main steps in the manufacture of vaccines.

The Post-Marketing Process

Figure 13 Decision timelines for capacity increases.

private markets; countries with different climates, epidemiological patterns, infrastructures, etc.; and, as a result, countries with different immunization schedules, vaccination coverage, and logistical requirements. Supplying vaccines, therefore, means being able to supply the right products with the appropriate presentation maintained in the cold chain—another vaccine specificity—in the right quantities and specifications, locally licensed and batch released, at an appropriate price, not to mention promotional activities. Obviously, for the reasons already described above, there is no substitute to proper forecasting and planning to face an ever-increasing demand. Even in industrialized countries, where normal market mechanisms operate, supplying a country’s vaccine needs remains a challenge and often requires an interactive partnership between public authorities and industry (43,44). The challenge is even greater for less developed countries, where the logistical infrastructure is often inadequate (with respect to both the physical distribution of vaccines and the administration of vaccines to target populations) and the buying power is low.

Obtaining the product license is far from being the last step in the regulatory life of a vaccine. Risk management, PMS, and pharmacovigilance are becoming increasingly important activities that need to be anticipated from the initial stages of product development. The concept of risk management (45) has recently emerged as a way to anticipate and manage actual or potential safety issues linked to the use of medicines. It is a global and continuing process aimed at detecting and minimizing risks throughout a product life cycle to optimize its benefit/risk balance from a patient safety end point. Risk management incorporates enhanced scientific expertise, specific methodological approaches, and multidisciplinary collaboration. Its application to vaccines and vaccination is critical because of their use as primary prevention measures in large healthy populations, including young infants. It raises very specific issues such as the benefit/risk ratio of the public health intervention. For instance, since 2005, risk management has been highly regulated in the European Union and each new license application must include a pharmacovigilance plan and risk minimization strategies. Implementing this global approach will not only justify the development of new competencies and tools but will also lead to a revisiting of existing company processes and organization. This is a business reality illustrated by drug/vaccine withdrawals, product liability, and litigations. Adverse events are indeed more and more often spotlighted as major public health issues, resulting in increasing regulatory demand. PMS implies a shared responsibility between many actors, not only the pharmaceutical industry and regulators, but also health care professionals, the exposed population, politicians, lawyers, etc. This will result in a large range of activities, including surveillance programs, procedures, monitoring tools, communication, etc. PMS/pharmacovigilance is essential for vaccines to identify possible risks that have not been detected in pre-licensure studies. These could be events with a low incidence or late occurrence, populations not studied, long-term follow-up of certain adverse events, or specific safety aspects, for example, those of combined vaccines or related to vaccine components.

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Finally, measuring the level of implementation and the impact of vaccination policies on the epidemiological behavior of the disease or the pathogen is also a critical part of PMS for vaccines. These processes are connected by a common thread, that is, regulation. Despite repeated promises of faster, simpler, more abbreviated regulatory requirements, the fact is that regulations have continued to increase in number and complexity at all stages of the vaccine development paradigm. Regulatory units are part of multidisciplinary pre-project assessments, as well playing an important role all along the development of the product, culminating in the licensing file; regulatory unit involvement continues post licensure after the vaccine is on the market. The new element that has emerged over the last decade has been an increased dialogue between industry and public regulatory bodies, which is making the overall process more predictable, albeit more complex and costly.

SOCIETAL ISSUES In a world where the ‘‘right to health care’’ is becoming an increasing concern, this situation raises issues of access, which some companies have addressed for a long time already, as well as issues of perception of vaccines.

Access to Immunization Intellectual Property Intellectual property (IP) is key to the survival of companies and to the pursuit of innovation (2,10,31). It helps assure them an acceptable return on their research activities (which includes both successful and unsuccessful projects). This translates into pricing policies that will provide a financial return for a period of time. Sometimes this leads to a situation where certain new vaccines prove to be too expensive for developing countries unless external financial assistance is provided. This may be true in principle, albeit not necessarily in practice. All EPI vaccines except hepatitis B have lost their patent protection a long time ago, and these vaccines (including Hepatitis B) have been sold at very low prices to public buyers in less developed countries. Still, quite often supply is insufficient because of limited production capacity and the low prices, not because of patents. In other instances, demand does not materialize because of the lack of immunization services infrastructures or health policies in the developing countries. The situation is obviously quite different for new patented products where the originating companies have to be guaranteed a fair return on their considerable investment. Maintaining or even reinforcing patent laws is a must in developed countries. This has taken place to some extent with the extension of patent laws in some countries, for new indications and for orphan products, where the very limited size of the potential market is a strong deterrent to industry’s interest. At the same time, patents have also been weakened in a number of ways: the early introduction of generics (through such mechanisms as ‘‘Bolar’’ in the United States), the large number of countries still not enforcing patent laws, and the threat of compulsory licensing not only in less developed countries but also sometimes in major markets (such was the case in the United States, linked to the bioterrorism scare). More generally, in the post-Seattle (antiglobalization movement), post-Pretoria (South African judicial ruling overriding patent), post-Doha circumstances, society at large has been applying

strong pressures to make pharmaceuticals and vaccines more readily available to the poorest countries. Industry, while fully aware of these changes, needs to understand where this is taking such things as IP protection, which remains the main stimulus to innovation. Creative thinking is needed to develop new ideas to ensure compatibility between these apparently conflicting goals (46–48). It will probably take time and a mix of different steps from all parties to reach a satisfactory solution, even though substantial progress has already taken place. As for generics, low-priced copies of vaccines could be seen as a way to level competition. But in practice, vaccines are very different from pharmaceuticals. A vaccine is licensed for manufacture in a given facility, and each batch has to be individually tested and released, whereas biologicals are not as strictly characterized as most pharmaceuticals. This makes it almost impossible for a true generic vaccine to exist, and the concept here is one more of ‘‘biosimilars’’ than of ‘‘generics.’’ Technology Transfers One of the ways to improve access for the less developed countries has sometimes been envisioned via transfers of technology. While these transfers may look like an easy solution, heretofore in practice this has certainly not been the case. Setting up a manufacturing facility acceptable to regulatory authorities is not an easy task. It requires a significant financial investment, adequately trained people, a suitable local environment (especially regulatory for controls), and support from other partners. It also requires a large enough guaranteed market and several years to come on line (46). As a result, a technology transfer can only succeed if it is very well planned, through a stepwise approach and a learning process (32). An ill-planned/ill-conceived technology transfer will only result in failure, frustration, and resentment. Also, such transfers will have to be financially successful. Obviously, if prices are too low, this will prove detrimental for local companies, which need to invest heavily to catch up with their counterparts in developed countries. With the emergence of viable, competitive local manufacturers in the major less developed countries such as India, Indonesia, Brazil, China, etc., the industrialized-country manufacturers have to decide whether to delocalize some productions or to enter in partnerships, joint ventures, or outright transfers of technology as an element of their long-term strategy (32). This has already taken place in a number of countries and will certainly develop further over the years. Dual Track Because of the vast needs and the strong downward pressures on vaccine prices for the poorest countries, practical solutions have had to be identified to ensure supply. Over time, this has developed into a situation where, for many vaccines, a ‘‘dual track’’ often exists between industrialized versus developing countries, for example, acellular pertussis versus whole-cell pertussis vaccines, Jeryl Lynn–based measles/mumps/rubella (MMR) versus monovalent measles, IPV versus OPV, combined versus monovalent vaccines, monodose versus multidose vaccines, thiomersal-free versus thiomersal-containing vaccines, and wide access versus limited access to new vaccines. Obviously, dual track has been closely related to dual pricing. Although ethically arguable, in principle, such a situation has proved to be a pragmatic answer to a difficult, complex situation. Taking a purely ideological stance can be counterproductive. What is probably a better answer is to make sure

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that any solution addresses epidemiological needs, is efficacious and safe, is implemented with quality standards at the same level as for industrialized countries, and is price competitive. Over time, the gap will have to be narrowed. Tiered Pricing The practice of differential prices between affluent and poor countries has existed for a long time, especially for vaccines, and mostly for companies of European origin. This is linked to many factors, including Europe’s historical relationship with these countries and different legal as well as cultural backgrounds between Europe and North America. This practice has been mostly applied to basic EPI vaccines for international tenders or local public markets (the most important ones) but much less for local private markets. Tiered pricing policies are only possible if they are limited to certain (the most needy) countries and if ‘‘normal’’ prices are implemented in developed markets, allowing not just paying for R&D, investments, margins, etc., but also—in a way—‘‘subsidizing’’ these lowerpriced markets. Such a strategy cannot be solely the responsibility of industry. Public authorities and international institutions and NGOs also have to be involved, recognizing some basic rules and guaranteeing, among other things, that no reimportation will occur back into developed markets, both for economic and safety reasons.

Perception of Vaccines Along with access, the perception of vaccines is one of the other important issues facing immunization policies and policymakers. Vaccines are given to healthy individuals to prevent disease. The public’s expectation of vaccines is that they should confer a high level of efficacy without causing significant side effects, a situation quite different from pharmaceuticals, which are mostly used to treat sick people. Because some vaccinepreventable diseases have almost disappeared from many industrialized countries, fear of the vaccine sometimes becomes more prevalent than the fear of the disease (49). There is a paradox in the claim that immunization is a ‘‘human right’’ but that getting immunized should result from an individual decision that respects ‘‘individual rights.’’ This is especially problematic for vaccines because high coverage is required to ensure effective ‘‘herd immunity’’ and, for some diseases, to allow regional elimination or even the graal of global eradication (e.g., polio). At a time when vaccines have led to the quasidisappearance of a number of diseases in many countries, the need to immunize is less strongly perceived and the side effects are more visible and widely discussed. Rare safety issues—sometimes based only on theory— can severely impact the success of immunization policies or campaigns in developed and less developed countries (49). In an era of instant and global communication (50), isolated incidents publicized by strong anti-vaccine lobbies can have devastating effects on public health. All parties, including industry, should accept that the paradigm has changed. Presently, the consumer and the regulator have assumed dominance, and the power is now largely in the hands of the consumer and the media (50). Also, recent developments such as the ‘‘principle of precaution’’ are here to stay. In such a situation, new attitudes need to be developed, accepting that all parties are accountable. We cannot just assume that vaccines are good for mankind and advocate their increased use. We also have to be more transparent, more informative, and more

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open to challenge; we have to implement active ‘‘vaccine vigilance’’ tools (49,51–53). We have to anticipate and more effectively coordinate efforts and be prepared to stand by scientific data while accepting that they may not always be the only answer to society’s concerns in today’s world.

CONCLUSION The future of immunization and vaccines is bright. Never has the field been as attractive, with new scientific advances, technical tools, medical understanding of diseases, and an increased recognition of the role of prevention and of economic and financial issues. Accordingly, industry is reinforcing its commitment to the field. While the opportunities are great for both public health and industry, nevertheless, the challenges are also multifold and substantial with the pace of scientific innovation, time, money, and know-how being the major constraints. Even if newcomers to industry become numerous in future, it remains to be seen how many will have the resources and strategic commitment to persevere and overcome the significant entry barriers, before they can compete across the board.

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36. Haase M. Regulating vaccines involving new technologies to ensure safety and timely licensure—the challenge. Vaccine 2001; 20(suppl 1):S68–S69. 37. Summary of the joint statement on thimerosal in vaccines. American Academy of Family Physicians, American Academy of Pediatrics, Advisory Committee on Immunization Practices, Public Health Service. MMWR Morb Mortal Wkly Rep 2000; 49(27):622, 631. 38. Van Hoof J. Manufacturing issues related to combining different antigens: an industry perspective. Clin Infect Dis 2001; 33(suppl 4): S346–S350. 39. Monahan TR. Vaccine industry perspective of current issues of good manufacturing practices regarding product inspections and stability testing. Clin Infect Dis 2001; 33(suppl 4):S356–S361. 40. Vose JR. Perspectives on the manufacture of combination vaccines. Clin Infect Dis 2001; 33(suppl 4):S334–S339. 41. Falk LA, Ball LK. Current status and future trends in vaccine regulation—USA. Vaccine 2001; 19(13/14):1567–1572. 42. Falk LA, Arciniega J, McVittie L. Manufacturing issues with combining different antigens: a regulatory perspective. Clin Infect Dis 2001; 33(suppl 4):S351–S355. 43. Poland GA, Marcuse EK. Vaccine availability in the US: problems and solutions. Nat Immunol 2004; 5(12):1195–1198. 44. Anonymous. Vaccines–endangered species? Nat Immunol 2002; 3 (8):695. 45. Hessel L. Risk management, vaccines and vaccination. Screening 2005; 4:18–20. 46. Batson A, Milstien JB. The GO License: only part of the solution. Health Aff (Millwood) 2008; 27(1):140–142. 47. Satyanarayana K, Srivastava S. Poverty, health & intellectual property rights with special reference to India. Indian J Med Res 2007; 126(4):390–406. 48. Dentico N, Ford N. The courage to change the rules: a proposal for an essential health R&D treaty. Plos Med 2005; 2(2):e14. 49. Chen RT. Vaccine risks: real, perceived and unknown. Vaccine 1999; 17(suppl 3):S41–S46. 50. Cookson C. Benefit and risk of vaccination as seen by the general public and the media. Vaccine 2001; 20(suppl 1):S85–S88. 51. Singleton JA, Lloyd JC, Mootrey GT, et al. An overview of the vaccine adverse event reporting system (VAERS) as a surveillance system. VAERS Working Group. Vaccine 1999; 17(22):2908–2917. 52. Andrews NJ. Statistical assessment of the association between vaccination and rare adverse events post-licensure. Vaccine 2001; 20(suppl 1):S49–S53. 53. Murphy TV, Gargiullo PM, Massoudi MS, et al. Intussusception among infants given an oral rotavirus vaccine. N Engl J Med 2001; 344(8):564–572.

9 Reaching Every Child: Achieving Equity in Global Immunization R. Bruce Aylward, and B. Melgaard Department of Vaccines and Biologicals, World Health Organization, Geneva, Switzerland

M. Birmingham World Health Organization Country Office, Bangkok, Thailand

J. Lloyd Program for Appropriate Technology in Health (PATH), Ferney-Voltaire, France

R. Eggers, J. Bilous, and T. Cherian Department of Immunizations, Vaccines, and Biologicals, World Health Organization, Geneva, Switzerland

INTRODUCTION Since the early 1960s, high quality vaccines have been available to counter many of the most common infectious diseases that kill or severely harm children, such as measles, diphtheria, pertussis, polio, and tetanus (1). Until as recently as 1975, however, less than 5% of the world’s children had access to these vaccines, despite immunization being the most costeffective of health interventions (2,3). That extraordinary progress has been made in rectifying the gap in childhood immunization coverage between rich and poor countries is primarily the result of one of the largest and most successful public health initiatives ever—the Expanded Program on Immunization (EPI). Through this program, a global network of national immunization services was developed, such that within 15 years, routine coverage of children had risen to nearly 75% worldwide (4), and world leaders were backing new and ambitious global immunization goals such as the eradication of poliomyelitis, and the elimination of measles and neonatal tetanus (NT) (5). Today, the EPI is making a major contribution to the achievement of the millennium development goals by reducing childhood mortality, and immunization rates are commonly used as a national development indicator. Although extraordinary progress has been made toward reaching all children with immunization services, in 2006, more than 20% of the children in the world were still not routinely receiving the basic childhood vaccines, and as a consequence, nearly 2 million children died of vaccine-preventable diseases, with the vast majority of these deaths occurring in developing countries (6). This chapter outlines the history and accomplishments of the global EPI effort and summarizes the challenges that must be overcome, particularly in developing countries, if every child is to be safely immunized with effective and appropriate vaccines as early as possible in life.

1974–1984: THE EPI IDEA AND THE EPI TOOLS Origins The success of the global smallpox eradication initiative was perhaps the most important stimulus for a WHO program to support the development of routine immunization services in developing countries (2). By the end of 1973, smallpox had been restricted to only five countries in Asia and Africa (7), and it was widely agreed that the momentum of the Intensified Smallpox Eradication Programme should be exploited to control other vaccine-preventable diseases (8). This consensus led WHO to establish the EPI in 1974, with the objective of raising childhood immunization coverage with an expanded number of antigens in an increasing number of countries (2). While the Intensified Smallpox Eradication Programme remained WHO’s highest immunization priority through the late 1970s, this period was used to develop the basic EPI principles such as the optimum vaccines and delivery strategies for developing countries. Though the EPI initiative arose out of the smallpox program, many important elements were to differ substantially, perhaps none more so than the strategic approach. For example, by the mid-1970s the smallpox eradication program had demonstrated the utility of wide-scale mass immunization campaigns to control other important vaccine-preventable diseases, such as measles, in developing countries (9). Following a large EPI feasibility study begun in Ghana in 1976, however, the founders of EPI opted to promote the delivery of vaccines through routine immunization services rather than large-scale campaigns. The original ‘‘EPI’’ vaccines were determined largely by the global relevance of the target disease, the availability of low-cost vaccines, and the cost-effectiveness of its control through immunization (10). Initially, antigens against six diseases were included BCG (against tuberculosis), DTP (against diphtheria, tetanus, pertussis), polio, and measles for infants, with tetanus toxoid for pregnant women to prevent neonatal

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Table 1 WHO-Recommended Infant EPI Schedule, 2004 Age Vaccine

Birth

BCG Oral polio DTP Hepatitis B—scheme Aa Hepatitis B—scheme Ba Haemophilus influenzae type b Yellow fever Measles

x x x

6 wk

10 wk

14 wk

x x x x x

x x

x x x x x

x x

9 mo

xb xc

a Scheme A is recommended in countries where perinatal transmission of hepatitis B is frequent (e.g., in Southeast Asia). Scheme B may be used in countries where perinatal transmission is less frequent (e.g., sub-Saharan Africa). b In countries where yellow fever poses a risk. c A second opportunity to receive a dose of measles vaccine should be provided for all children. This may be done either as part of a routine schedule (if very high routine coverage is reached regularly) or in a campaign.

tetanus. As additional vaccines were recommended for universal introduction, the immunization schedule for infants was adapted (Table 1) (11). Over time, specific immunization schedules and policies, often quite different from those of industrialized countries (i.e., younger ages of administration), were established to optimize the uptake and impact of these vaccines in the developing country setting (12–14). It was in 1977, the year of the last case of smallpox (7), that the World Health Assembly (WHA) formally declared the EPI goal of delivering these six antigens to the world’s children by 1990 (15). It is difficult to overstate the obstacles that existed in the late 1970s between the manufacture of a vaccine, usually in an industrialized country, and its safe administration under field conditions in a developing country, thousands of miles away (16). Despite the tremendous diversity of the countries and cultures targeted by the EPI, in these early years, a standardized approach to overcoming the huge operational and technical barriers to universal childhood immunization was to prove feasible. The first EPI operations manual was developed by 1977, with seminars conducted in most of the six WHO regions to introduce the program to senior public health officials (2). These seminars established agreement on many critical issues including the standard EPI immunization schedule, a global EPI position on contraindications to specific vaccines, and the first standardized ‘‘global’’ reporting system for immunization. Most importantly, these training seminars emphasized the application of management principles to EPI.

Equipment Essential to moving from guidelines to actual immunization was the development and distribution of a range of cold-chain equipment that could maintain the potency of the EPI vaccines while withstanding the rigorous environment and demanding conditions of developing-country use, particularly in rural tropical areas. Although the stability of the EPI-recommended vaccines varied depending on the antigen, the thermolability of oral polio vaccine (OPV) and the sensitivity to freezing of DTP dictated the parameters for the cold chain (originally set at 0–88C, these were raised to 2–88C in the year 2000 as newer, more expensive, and more freeze-sensitive vaccines were added) (17,18).

Figure 1 Solar refrigeration equipment being installed in a health facility. Source: Courtesy of Program for Appropriate Technology in Health (PATH).

Because very little equipment that could guarantee these conditions amid the high or very low ambient temperatures and unreliable electricity supply in many developing countries was available, by the late 1970s, WHO’s EPI had begun working with manufacturers to produce low-cost equipment for storing and transporting vaccines (19). The technological solutions that were found included ice-lined refrigerators designed to protect vaccines against interruptions in the electricity supply, and small, robust refrigerators for remote health centers operating on kerosene, gas, and solar energy (20,21) (Fig. 1). By 1979, WHO had established a network of laboratories to evaluate new equipment, and had published the first edition of the WHO/ UNICEF product information sheets (PISs) (22), detailing the immunization equipment that met WHO specifications, and could be recommended for use in developing countries. The PIS has subsequently been replaced with a web-based Performance, Quality and Safety (PQS) prequalified device and equipment, as a continued essential resource for developing country immunization programs, covering a wide range of cold chain, injection, disposal, and other equipment (23). At the same time that this capacity to deliver potent vaccines to the field was being established, it was also critical to minimize the risk of infectious complications due to unsterile EPI injections (24). This has required the continuous development and evaluation of new injection, sterilization and disposal

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In 1978, national EPI reviews were introduced using a soonto-be standardized methodology and joint national/international teams to evaluate all aspects of the immunization operations from the central to peripheral levels. Three hundred such reviews were conducted by 1994, often including a ‘‘30-Cluster EPI Coverage Survey,’’ using the methodology that WHO had developed to corroborate reported immunization coverage with a basic coverage survey (31,32). These reviews played an important role in the development of national programs by motivating staff, identifying key areas for improvement, and raising political support by formally presenting the recommendations to national health authorities.

1985–2000: OPTIMIZING THE IMPACT OF EPI Expanding Access Figure 2 An autodisabled syringe. Source: Courtesy of Program for Appropriate Technology in Health (PATH).

equipment, and strategies (25,26). In 1984, for example, the original glass syringes and open boiling equipment of EPI were replaced with sterilizable syringes and portable steam sterilizers, which had been developed specifically for WHO for use in field conditions. As understanding of the risks posed by injections and injection equipment accumulated, EPI policy shifted toward the use of disposable injection equipment. To protect against the potential reuse of this equipment, WHO facilitated the development of the ‘‘auto-disable syringe’’ that could not be used more than once. This type of syringe, first used in mass vaccination campaigns, is now an essential component of a universal policy of WHO and UNICEF to use only auto-disabling injection equipment for all immunizations (Fig. 2) (27,28).

Implementation, Evaluation, and Oversight To extend immunization services beyond urban centers, it was necessary to teach immunization techniques, injection safety, cold-chain maintenance, and program management from the national to village levels. By the end of 1977, the original EPI operations manual had been expanded into a training course for senior-level immunization program managers, and the first course held in Kuala Lumpur, with participants from 19 countries. These materials were soon supplemented with training courses for cold chain and logistics (1978) and training and supervision (1979) (29). By 1982, over 9500 people from 83 countries had been trained in one or more of these courses (2). These standard training materials have continuously been updated and expanded to include topics that now range from refrigerator repair to motorcycle instruction. The routine monitoring of global immunization performance by the WHO began in 1977. A central EPI information system was developed, with computer software for monitoring, at the national and regional levels, immunization coverage, surveillance data, and cold-chain equipment. The data generated permitted the first systematic estimates of developing country immunization coverage, thus facilitating the targeting of international technical and donor assistance. National immunization programs now annually report at all levels from the service delivery to the national level routine immunization coverage, surveillance, and other program data to WHO and UNICEF through a standardized reporting process (30).

Although the necessary EPI policies, strategic approaches, and equipment were largely in place by 1984, less than 50% of the world’s children were being ‘‘fully immunized’’ at that time (2). Achieving the goal of universal childhood immunization by 1990, defined as 80% coverage in the WHA Resolution of 1977, would require a massive acceleration of activities. Recognizing this, the Rockefeller Foundation hosted a high-level meeting of potential EPI supporters and partners, cosponsored by the WHO, UNICEF, the World Bank, and the United Nations Development Program (UNDP) in 1984 in Bellagio, Italy (33). The 34 leaders in attendance—from developing countries, public health institutions, and international agencies—strongly endorsed the EPI concept and committed to its further expansion. To facilitate the coordination of international assistance to national immunization efforts, the cosponsors of the meeting established the Task Force for Child Survival and Development. UNICEF played a particularly critical role in the acceleration of immunization activities. Its charismatic leader, James Grant, adopted the goal of universal childhood immunization (UCI) and vigorously promoted it with national leaders (34). UNICEF further helped support national immunization programs by operationalizing the EPI through its regional and country offices. In addition to UNICEF’s early role in the international procurement of WHO-approved vaccines and equipment, the organization also played a major role in the national social mobilization and communications efforts that were to improve community awareness of, and demand for, routine immunization services. With the expansion of immunization activities, new technical issues arose and often threatened to undermine the optimal implementation of the programs. Ongoing operational and epidemiological research was essential to resolve vaccine or immunization issues that were often unique to developing countries (2). For example, although the epidemiology of the EPI target diseases was well understood in industrialized nations, the burden of disease because of poliomyelitis and neonatal tetanus was often grossly underestimated in developing countries, frequently holding up the introduction of one of those vaccines in a particular country. These hurdles were only overcome after the development and implementation of standardized lameness and neonatal mortality surveys, demonstrating the importance of these diseases in the developing country setting (35,36). Similarly, specific policies were needed to counter misconceptions about contraindications to EPI vaccines and reflect the safety of their simultaneous administration (37–39).

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Figure 3 Global immunization DTP3 coverage based on official reports from countries to WHO, 1980 to 2006. Source: WHO/UNICEF coverage estimates 1980 to 2006, August 2007 193 WHO Member States.

As the technical, financial, political, and logistical challenges to reaching all children with the EPI vaccines were systematically addressed, the period 1985 to 1990 saw a tremendous rise in routine immunization activities worldwide. By 1990, vaccines were protecting nearly 75% of the world’s children from measles, tetanus, polio, diphtheria, and pertussis. In the 1990s, a period of stagnation in immunization coverage followed, which was slowly overcome in the first years of the new millennium (Fig. 3).

Accelerating Disease Control As national immunization services were established or strengthened in developing countries, from the late 1980s onward WHO’s technical oversight body for EPI turned its attention to the accelerated control of key EPI target diseases. In doing so, EPI was in fact returning to its original mandate in that ‘‘the primary concern of EPI is not immunization but disease control, using immunization as the strategy’’ (40). This shift in emphasis was motivated by a number of other factors, not the least of which was the need to maintain donor and political support for what had become a very successful public health program. Consequently, in 1989 new EPI goals were established that went beyond the raising of routine immunization coverage to include the eradication of poliomyelitis, the elimination of neonatal tetanus (NT), and the reduction of measles mortality and morbidity by 90% and 95%, respectively (41). The international political importance of these ambitious goals increased substantially in 1990 when they were endorsed at the World Summit for Children, the largest ever gathering of heads of states (5). Despite this early attention, outside of the American and Western Pacific regions, there was limited progress toward any of these goals prior to the mid-1990s. The main reason for this was

the widespread deterioration in the quality, coverage, and commitment to routine immunization that had begun in the early 1990s because of a number of factors (42). Of particular importance, the rapid gains of the EPI expansion in the late 1980s appears to have fuelled among donors a false sense of the robustness of the program, leading to a rapid contraction of international financial support, usually before other more sustainable funding had been secured (43–45). Around the same time, structural adjustment programs began to markedly affect national budgets and staffing patterns in many developing countries. These problems were further compounded by health sector reform processes, which frequently led to a stagnation of EPI performance, as the highly centralized EPI structures were integrated with other child health services and/or critical functions and the staff were devolved to the subnational level (46). Even within the UN agencies, the growing demands of other public health priorities and programs limited the time, attention, and resources that staff of all levels could devote to immunization. By 1995, however, efforts to achieve the specific diseasecontrol goals of EPI had stimulated the development of new strategic approaches for reaching every child, including close collaboration with many new partners. In particular, the goal of global polio eradication had a massive impact, as it grew into the largest public health initiative ever. Spearheaded by WHO, Rotary International, UNICEF, and the U.S. Centers for Disease Control and Prevention (CDC), the goal of polio eradication brought together a broad coalition of donor and technical partners to support national efforts to improve the reach of immunization services and establish effective surveillance. Through a combination of routine immunization, national immunization days (mass vaccination campaigns), surveillance for acute flaccid paralysis (AFP), and house-to-house mop-up activities, polio fell from an

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Figure 4 Estimated annual number of measles deaths in the WHO African region, all ages, 2000 to 2006. Source: WHO/IVB measles deaths estimates, November 2007.

estimate of more than 350,000 cases (35,000 reported) in over 125 countries in 1988 to 471 cases in 10 countries in 2007 (as at September 12, 2007) (O. Rosenbauer, Global Polio Eradication Initiative, WHO, personal communication). Substantial progress toward the measles control goals was first made in the Americas by building on the infrastructure and strategic approaches that had been established for polio eradication. In 1985, the WHO region of the Americas went beyond the original goal, adopting a resolution to eliminate measles in all

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member states. By conducting massive ‘‘catch-up’’ campaigns, raising routine immunization coverage, introducing case-based measles surveillance, and establishing regular ‘‘follow-up’’ campaigns, indigenous measles was virtually eliminated from the region (47,48). By end of 2000, a global action plan for accelerated measles control had been developed, four WHO regions had established measles elimination goals, and a wide variety of countries in all regions had implemented the Pan American Health Organization (PAHO) strategic approach (49). Considerable progress was made globally in the overall reduction of measles mortality, and the 2005 measles mortality reduction goal was met (50,51) and then surpassed in Africa (Fig. 4). Perhaps the most challenging of these goals was NT elimination, defined as less than 1 case per 1000 live births in every district of every country. The disease had little visibility as onset occurred very early in life, and predominantly in rural areas among underprivileged populations. Furthermore, prevention required the immunization of women, rather than children, with multiple doses of tetanus toxoid during antenatal visits. Worldwide mortality from neonatal tetanus was estimated at 180,000 in 2002, which represents a 78% reduction since the late 1980s (52). Consequently, in November 2000, a new fiveyear strategic plan was launched to achieve the goal by 2005, through routine and supplemental TT vaccination, strengthening of clean birth delivery services, and surveillance to detect and target areas and populations at high risk (53). By end-2001, 20 of the 57 targeted countries had plans of action and 13 were already implementing activities, and by 2007, 110 of the developing countries had completed the process under which the elimination of maternal and neonatal tetanus was verified (Fig. 5). Using a ‘‘lot quality assessment’’ methodology (54),

Figure 5 Maternal and neonatal tetanus elimination status, 2006. Source: WHO/IVB database, 2007, 193 WHO member states; data as of August 2007.

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these countries demonstrated that the districts with the highest likelihood of not having eliminated NT, had in fact fewer cases than the elimination threshold. The pursuit of these accelerated disease control goals has helped raise the political visibility and attention to childhood immunization, particularly as national leaders personally launched the massive immunization campaigns. These initiatives also greatly enhanced the international investment in global disease surveillance and developing country immunization programs. By the year 2006, for example, WHO alone had deployed over 3300 national and international personnel through the polio initiative to provide technical and administrative support to national immunization programs worldwide. New, efficient partnerships for immunization were also established through these initiatives, governed by cross-agency strategic plans and coordinated through national or regional level ‘‘interagency coordinating committees’’ (ICCs). Perhaps most importantly, these efforts resulted in regular access to previously unreached geographic areas and populations, particularly through the strategic approaches and community engagement of the polio eradication initiative. In 2006, more than 375 million children were reached with multiple doses of OPV, during 187 immunization campaigns across 36 countries, with 2.1 billion doses of OPV (55).

Introducing New Antigens and Interventions As the expansion of the global EPI network significantly enhanced access to children worldwide in the late 1980s, increasing attention was given to the feasibility of systematically using the EPI infrastructure to deliver other antigens and, potentially, other health interventions. Despite the logic and cost-effectiveness of many such proposals, it was not until 1999 that substantial progress was made in this regard. The first additional antigen to be formally recommended for inclusion in EPI since 1974 was the yellow fever vaccine in 1989 (56). Recognizing the resurgence of yellow fever as a public health problem in Africa in the late 1980s, in 1989, WHO’s EPI technical oversight body recommended that 33 countries on the African continent include this vaccine in their infant immunization programs. By 1999, however, reported yellow fever vaccine coverage in these countries was only 19%, compared with 80% for measles, which is given at the same age (9 months). The reasons for this poor uptake varied by country, ranging from a lack of resources for vaccine purchase to a failure to appropriately modify or implement national immunization policies. The first ‘‘new’’ vaccine to be included in EPI was the hepatitis B vaccine. Though licensed in 1981, its very high cost initially prohibited wide-scale use, even within industrialized countries. Even when the cost began to decline, however, there was limited support for universal childhood immunization against hepatitis B in some international health circles. The reason is that the disease did not cause substantial childhood morbidity, even though it was a leading cause of liver cancer and early death among males in developing countries. By the late 1980s, the price of the vaccine began to decline as new producers emerged and the market expanded. In 1992, further declines in the vaccine price, combined with increasing cost-effectiveness data and the failure of targeted hepatitis B immunization strategies, warranted WHO’s recommendation for universal childhood immunization (57,58). By 1999, 85 WHO member states, primarily high and middle income, had implemented the

recommendation, but the vaccine remained unavailable in most of the poorest countries, which harbored the highest burden of the disease (59). Despite compelling data as to the appropriateness of including yellow fever and hepatitis B vaccines in EPI, it was not until after 1999 that there was the opportunity to substantially improve the uptake of both vaccines in the world’s poorest countries. Of the non-vaccine interventions promoted for inclusion in routine immunization services, the most important has been vitamin A. Since the discovery in the 1970s and 1980s that regular supplementation with that micronutrient could reduce all cause childhood mortality by as much as 23% (60), there has been substantial interest in administering it during routine immunization contacts, particularly with measles at nine months of age (61). Although the logistical implications of including vitamin A are relatively minor, as it is heat-stable and administered by mouth, by 10 years after the recommendation for its routine use in conjunction with immunization services, only a limited number of countries had implemented the policy. In the late 1990s, however, the linking of vitamin A and immunization contacts took a major step forward when countries began including the micronutrient in polio national immunization days (NIDs) (62). By 2000, there were compelling data to further promote the use of vitamin A during routine immunization contacts—inclusion of the micronutrient in polio NIDs in 61 countries in the period 1998 to 1999 had averted over 400,000 childhood deaths (63). By the end of 2000, 49 of the 136 countries with known vitamin A deficiency had begun distributing the micronutrient through routine immunization services. Recognizing the opportunity that the EPI infrastructure provided, and building on the experience gained through the introduction of the yellow fever and hepatitis B vaccines and vitamin A, in the early 1990s WHO developed a framework to facilitate the introduction of new interventions (13). This framework is intended to accelerate the evaluation of an intervention and, if necessary and feasible, modify its characteristics to streamline its integration into EPI.

2001–2007: A NEW MILLENNIUM AND THE GLOBAL IMMUNIZATION VISION AND STRATEGY In the year 2000, ambitious millennium development goals were underwritten by all nations. These goals include a two-thirds reduction in child mortality by 2015. For this to be achieved, the full potential of the global EPI network must be exploited to prevent the 1.4 million vaccine-preventable childhood deaths that continue to occur every year (Fig. 6). Preventing these deaths requires extending the reach of immunization services to the more than 25% of the world’s children who are yet to have regular access to immunization, the most cost-effective of health interventions. Particular attention must be given to increasing coverage among the world’s poorest children who are disproportionately affected by vaccine-preventable and infectious diseases. That reaching all children with immunization is a feasible goal is abundantly evident in the progress achieved and experience gained over the past 25 years. With the increased attention and funding for immunization, mainly through the Global Alliance for Vaccines and Immunization (GAVI) and a realization that immunization— both current and future vaccines—would play a major role in

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Figure 6 Distribution of the estimated 1.4 million annual childhood deaths from diseases that are preventable by routine childhood vaccination, 2002. Source: WHO/IVB estimates, 2004, adapted from World Health Report 2002.

reaching the millennium development goals for child survival, WHO and UNICEF in 2004/2005 developed a strategic document, the Global Immunization Vision and Strategy (GIVS) (64), which outlined the overall vision, direction, and core strategies. This document clearly transcends the realm of an organizational strategic plan of WHO or UNICEF—instead it outlines in broad strategic terms the direction that WHO and UNICEF believe immunization programs of the world and their partners should take in the period from 2006 to 2015, and how immunization will contribute to the reaching of the millennium development goals. GIVS set new global goals in immunization by 2010; for routine vaccination, it requires that all countries should reach 90% of their children with vaccines, and in terms of measles control, it dictates that mortality should be reduced by 90% compared to the 2000 level. The document describes four key strategic areas that would allow immunization programs to grow further. The first strategic area ‘‘protecting more people in a changing world’’ outlines strategies to expand the reach of vaccinations both geographically (by targeting the hard-toreach) populations and in terms of age groups, requiring the expansion of vaccination programs to reach children beyond the first year of life. The second strategic area ‘‘introducing new vaccines and technologies’’ describes the strategies needed to support countries to make the decision, and to implement new vaccine or technology introduction. In the third strategic area ‘‘integrating immunization, other linked health interventions, and surveillance in the health systems context,’’ the need for immunization programs to work in coordinated and integrated fashion with other programs, and the basic health system itself is outlined. This area also contains the key strategies required to broaden and strengthen the surveillance and monitoring systems necessary to run a successful public health program. Finally, in recognition that the immunization program operates in a global context of interdependency, the fourth strategic area ‘‘immunizing in the context of global interdependence’’ describes the necessity of sustainable financing and supply of

vaccines of assured quality, as well as communication, information dissemination, partnerships, and global epidemic preparedness. GIVS was welcomed by the WHA, and supported by a resolution in 2005 (65). The vision and strategy have been globally accepted by immunization partners and country programs as the common basis for coordinated implementation and donor support. Further strategic documents have resulted from the GIVS direction, including the Global Framework for Immunization Monitoring and Surveillance (GFIMS) (66), focusing on the strategic need for vaccine-preventable-disease surveillance and program monitoring to underpin GIVS.

NEW OPPORTUNITIES AND CHALLENGES Improving Access to Immunization In response to the inequities in access to immunization, especially where immunization coverage was less than 50%, in 2002, WHO, UNICEF, and other partners developed the Reaching Every District (RED) strategy, which described five operational components to be included in district immunization microplans to increase access. The five components of RED are reestablishing outreach, supportive supervision, linking services with the community, monitoring and using data for action, and planning and management of resources. The RED strategy encourages countries to prioritize districts with poor access and utilization of immunization, and then make microplans to identify local problems and adopt corrective solutions. Since 2003, 53 developing countries have started implementing RED to various degrees, mostly in Africa and South- and Southeast Asia. Data available in 2006 show that the impact of RED is mostly in the weakest districts, indicating that where RED is implemented, it can help to reduce gaps in immunization coverage, and in particular to bridge inequalities between districts. In many countries, outreach services, one of the five components of RED, were often used to deliver other interventions beyond immunization, such as vitamin A,

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anti-helminthic drugs, or insecticide-treated bed nets. This indicates that implementation of the RED strategy may have an impact beyond immunization services alone.

Introducing New Life-Saving Vaccines In one of the most important developments in the history of EPI, the GAVI was established in 2000, bringing new cooperation, resources, and tools to the strengthening of routine immunization programs, and introduction of new vaccines. Among the most important of the tools available to GAVI is the GAVI Fund (previously known as the Vaccine Fund), initially established with a US$ 750 million donation by the Bill and Melinda Gates Foundation, and since augmented by other foundations and donor governments such that by end-2006 it had received cumulative donations of US$ 1.8 billion since its inception (67). By the end of 2006, the GAVI boards had endorsed of a cumulative total of more than US$ 645 million for new and underused vaccines, US$ 202 million for immunization services strengthening, and US$ 106 million for injection safety support directly to countries. As of December 2006, the GAVI boards had already provided 61 countries with the financing necessary to introduce hepatitis B into their routine immunization services and 23 countries had received support for yellow fever vaccine purchase (68). The GAVI Fund had also facilitated the purchase of Haemophilus influenzae type b (Hib) vaccine for 24 countries. More recently, in December 2006, GAVI approved the investment cases for the country applications to introduce pneumococcal and rotavirus vaccines. The first country applications for these new vaccines were approved in October 2007, heralding a new epoch of newer vaccines. In addition to this support for new vaccines, the creation of GAVI has had an enormous impact on national and international efforts to revitalize immunization services through its high level political advocacy, interagency coordination, and, through the GAVI Fund, routine immunization financing. Insufficient financing remains a major barrier toward equity in immunization, and the search for sustainable EPI funding remains a chronic challenge for the world’s poorest countries. However, at no point in the history of EPI has there been as much attention to this area and as many promising initiatives. For example, countries receiving funds from the GAVI Fund are required to present a comprehensive and costed multiyear strategic plan (cMYP) that includes detailed costing and financing information, allowing the financial sustainability and the long-term needs of the country to be determined. Meanwhile, most countries are expanding the scope of the ICC, which was established to coordinate polio eradication partner inputs, to encompass all immunization activities. Also at the country level, the decentralization of health services, with the devolution of planning functions and budget allocations, provides opportunities to improve coordination with other sectors, and more effectively use resources to reach more children. Though the debt relief initiative for the highly indebted poor countries (HIPC) has yet to promote highly cost-effective health interventions such as immunization to the degree anticipated, this remains a potential mechanism for substantially enhancing financial support to immunization. At the international level, a GAVI task force is specifically addressing the issue of sustainable immunization financing, with a particular emphasis on understanding and exploiting the many opportunities that exist. Finally, the GAVI Fund has been extended and recapitalized for the period from 2005 to 2015, and it has

become one of the most important new immunization financing instruments for poor countries. To assist countries in these efforts, the UN agencies and immunization partners will need to provide technical and financial support at least equal to that which it has provided in the ongoing effort to eradicate polio. WHO and UNICEF have already detailed the major elements of the polio eradication infrastructure and begun planning the systematic transition of the institutional arrangements (e.g., ICCs, laboratory networks, technical oversight groups), physical infrastructure (e.g., cold chain, communications, and transportation equipment), and human resources to support broader immunization goals. In addition to the obvious logistical and managerial challenges to reach every child and ensure sustainable financing, the maturation of EPI has brought other extremely important though much less visible concerns. The importance of ensuring the safety of all immunizations led WHO to establish in 1999 a ‘‘priority project’’ in this area (69,70). This has brought together the broad range of ongoing work in this area from ensuring ‘‘vaccines of assured quality’’ [including functioning national regulatory authorities (NRAs) worldwide], to improving injection safety and the monitoring and management of adverse events following immunization (71,72). One of the most important developments in this project was the establishment of a WHO global advisory committee on vaccine safety (GACVS) to ensure the capacity to respond promptly, objectively, and with scientific rigor to vaccine safety issues anywhere in the world (73,74). One of the more insidious threats to achieving and sustaining equity in global immunization is the growing challenge of ‘‘vaccine security.’’ Ironically, the growth in global immunization activity and coverage has recently been accompanied by a contraction in both the number of manufacturers producing vaccines for the developing country market and the amount of vaccine they produce (75). The principal causes of this problem have been the consolidation of the pharmaceutical industry in the mid-1990s and the divergence of developing and industrialized country vaccine markets. Though UNICEF procured vaccines for 40% of the world’s children in over 100 countries in 2001, it is operating in an increasingly challenging market where the gap between demand and supply has narrowed substantially. To manage this challenge and stabilize the market, UNICEF and WHO have substantially enhanced longterm vaccine demand forecasting capacity, improved their knowledge of the vaccine market, increased dialogue with manufacturers, explored arrangements for longer-term funding and contracts for vaccines, and promoted sustainable financing mechanisms. The challenge will be to exploit the opportunities that exist to establish true equity in global childhood immunization.

CONCLUSION Since the inception of the EPI in 1974, global immunization coverage has risen from less than 5% to over 79% for DTP3 in 2006 (30). Today, immunization strategies have proved to be highly effective in reducing disease, national vaccine cold chain and logistics systems can reach even remote areas, and health staff have become skilled and experienced with operational issues. Wide reach operational guidelines and training materials are widely available.

Chapter 9: Achieving Equity in Global Immunization

Polio is now close to being eradicated, measles mortality has been reduced by more than half, and NT a public health problem in only a few countries. These achievements demonstrate that with the appropriate mix of planning, partnerships, resources, and community engagement, all children, everywhere can be regularly accessed with immunization services. The launch of GAVI in the year 2000 provided a mechanism to effectively tackle the financing gap that had become one of the greatest barriers to achieving equity in the introduction of new vaccines, and expanding services to the hardest to reach children. Finally, the wide-scale delivery of vitamin A, interventions against malaria, and anti-helminthic drugs during immunization contacts have begun to demonstrate the broader promise of the global EPI infrastructure to strengthen health systems. The capacity needed to achieve equity in the delivery of childhood immunization services on a global scale has never been stronger. The potential for immunization to reduce child mortality is greater than ever, but making use of this potential will depend on strong national ownership and international partnership, especially in cofinancing. There has been much progress in the EPI since 1974, but achieving equity in global immunization still requires sustained national commitment to identify those communities in most need, and overcome national obstacles to reaching every child.

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55. World Health Organization. Global Polio Eradication Initiative Annual Report 2006. Geneva: World Health Organization, 2006 (WHO document number WHO/Polio/07.02). 56. Joint UNICEF/WHO Technical Group on Immunization for the African Region. Report of the Third Meeting, Nairobi, 1988. 57. World Health Assembly. Immunization and Vaccine Quality. Geneva: World Health Organization, 1992 (WHA Resolution No. WHA45.17). 58. Vryheid RE, Kane MA, Muller N, et al. Infant and adolescent hepatitis B immunization up to 1999: a global overview. Vaccine 2000; 19(9–10):1026–1037. 59. World Health Organization. Vaccine-Preventable Diseases: Monitoring System—2000 Global Summary. Geneva: World Health Organization, 2000 (WHO Document No. WHO/V&B/00.32). 60. Beaton GH, Martorell R, Aronson KA, et al. Effectiveness of Vitamin A Supplementation in the Control of Young Child Morbidity and Mortality in Developing Countries. New York, NY: United Nations, 1993. ACC/SCN State-of-the-Art Series, Nutrition Discussion Paper No. 13. 61. Expanded Programme on Immunization. Global Advisory Group. Wkly Epidemiol Rec 1987; 62:5–12. 62. Goodman T, Dalmiya N, de Benoist B, et al. Polio as a platform: using national immunization days to deliver vitamin A supplements. Bull World Health Organ 2000; 78(3):305–314. 63. Ching P, Birmingham M, Sutter R, et al. The impact of vitamin A supplements delivered with immunization campaigns. Am J Public Health 2000; 90:1526–1529. 64. World Health Organization and the United Nations Children’s Fund. Global Immunization Vision and Strategy, 2006–2015. Geneva: World Health Organization, 2005 (WHO Document No. WHO/IVB/05.05). 65. World Health Assembly. Expanded Programme on Immunization. Geneva: World Health Organization, 2005 (Resolution WHA58.15). 66. World Health Organization. Global Framework for Immunization Monitoring and Surveillance. Geneva: World Health Organization, 2007 (WHO Document No. WHO/IVB/07.06). 67. Global Alliance for Vaccines and Immunization, GAVI. GAVI Alliance Progress Report 2006. 68. Global Alliance for Vaccines and Immunization, GAVI. GAVI Factsheets: Progress and achievements 2006. 69. Scholtz M, Duclos P. Immunization safety: a global priority. Bull World Health Organ 2000; 78(2):153–154. 70. Duclos P, Hofmann C-A. Immunization safety: a priority of the World Health Organization’s Department of Vaccines and Biologicals. Drug Saf 2001; 24(15):1105–1112. 71. Milstien J, Dellepiane N, Lambert S, et al. Vaccine quality–can a single standard be defined? Vaccine 2001; 2956:1–4. 72. Mehta U, Milstien JB, Duclos P, et al. Developing a national system for dealing with adverse events following immunization. Bull World Health Organ 2000; 78(2):170–177. 73. World Health Organization. Global Advisory Committee on Vaccine Safety. Available at: http://www.who.int/vaccine_ safety/about/indepth/en/index3.html. 74. World Health Organization. Report of the Global Advisory Committee on Vaccine Safety Meeting, 12 to 13 June 2007. Geneva: World Health Organization, 2007. Wkly Epidemiol Rec 20 2007; Vol 82, Issue 28/29:252–259. 75. United Nations Children’s Fund. Vaccine Security: Ensuring a Sustained, Ininterrupted Supply of Affordable Vaccines. New York: United Nations Children’s Fund, 2001 (UNICEF Document No. E/ICEF/2002/6).

10 A Paradigm for International Cooperation: The GAVI Alliance Lisa Jacobs GAVI Secretariat, Geneva, Switzerland

INTRODUCTION For more than a century, scientists and public health leaders have known that preventing infectious diseases is the most efficient form of health intervention. During the 20th century, medical research led to the development of vaccines that prevent a number of crippling, often fatal, childhood diseases. In fact, vaccines helped to reduce the health gap between rich and poor countries. Up until the 1970s, outside of the world’s richest countries most children did not get vaccinated against even a single disease. Following the successful eradication of smallpox in 1977, public health advocates and experts around the world collaborated to help build systems in developing countries to routinely provide infants with vaccination against six diseases—measles, diphtheria, pertussis, tetanus, poliomyelitis, and tuberculosis (using bacille Calmette-Gue´rin vaccine). By 1990, 75% of the world’s children received these ‘‘basic six’’ vaccines. In the history of international public health, there has been no other routine health intervention that has received such high coverage as infant vaccination. However, as a new century begins, the world falls short of realizing the full benefit of childhood immunization. By the end of the 1990s, approximately 34 million children were born every year that would not become immunized. In sub-Saharan Africa fewer than half of the children were being immunized. As a result, every year, approximately three million additional lives could be saved from easily prevented infectious diseases if vaccines could reach their target populations. Moreover, vaccines such as those against hepatitis B and Haemophilus influenzae type b (Hib) have not been introduced quickly enough in the poorest countries. New vaccines at late stages of development, such as those being created against pneumococcal pneumonia, meningococcal meningitis, and rotavirus diarrhea—diseases that kill millions of children every year in the developing world—are at risk of not reaching those who need them most. Finally, the search for vaccines against several of the most critical infectious disease threats of our time—HIV/AIDS, malaria, and tuberculosis—must be intensified, and effective health delivery systems must be strengthened to ensure that once these vaccines are successfully developed, all those in need can access them.

GAVI COMES ON THE SCENE The Global Alliance for Vaccines and Immunization (GAVI) was launched in 2000 as a coalition committed to reinvigorating and sustaining the promise of widespread immunization

(Box 1). A partnership that includes national governments, UNICEF, WHO, The World Bank Group, the Bill & Melinda Gates Foundation, nongovernmental organizations (NGOs), the vaccine industry, and research and technical health institutions, GAVI exists as a mechanism for coordinating and revitalizing immunization programs at international, regional, and national levels. GAVI Alliance mission statement: To save children’s lives and protect people’s health by increasing access to immunization in poor countries. GAVI Alliance strategic goals:

1. 2. 3. 4.

Contribute to strengthening the capacity of the health system to deliver immunization and other health services in a sustainable manner. Accelerate the uptake and use of underused and new vaccines and associated technologies and improve vaccine supply security. Increase the predictability and sustainability of longterm financing for national immunization programs. Increase and assess the added value of GAVI as a public-private global health partnership through improved efficiency, increased advocacy, and continued innovation.

Developing country governments have provided childhood immunizations as part of their health services for decades. In most cases the Alliance taps into existing capabilities and networks—including the staff and technical resources of partners—so there is no need to build brand new systems. WHO and other technical partners have been providing governments technical support to shape country programs and monitor results, and they continue to do so; UNICEF purchases most vaccines used by the poorest countries and it procures on behalf of GAVI. Finally, in-country collaboration teams had been set up in many countries for polio eradication; these are now used for GAVI-related issues. By significantly expanding the reach and effectiveness of immunization programs, country by country, the GAVI partners hope to decrease the burden of disease globally. GAVI reaffirms that immunization is a cornerstone for health, a key component of the broader framework of economic development and poverty reduction, and an essential step to protecting

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children’s health and allowing each child to reach his or her greatest physical and intellectual potential.

TYPES OF GAVI SUPPORT New and underused vaccines support (NVS)

FINANCIAL SUPPORT TO IMPROVE IMMUNIZATION The GAVI Alliance created the GAVI Fund, a financing mechanism designed to help achieve its objectives by raising new resources and swiftly channeling them to developing countries. Contributions from 16 governments, the European Union, The Bill & Melinda Gates Foundation, and private contributors make up the Fund. Additionally, the introduction of a new and unique funding mechanism in 2006, the International Finance Facility for Immunization (IFFIm), has resulted in dramatic increases in resources. The IFFIm is a new financing institution that uses pledges of future aid to raise money from international capital markets for immediate use. This breakthrough in development financing has radically altered the scale and timeframe of GAVI’s operations. The GAVI partners designed its financing instruments not to supplant other sources of funding but as a catalyst for additional funding. In fact, this has happened in many countries, with partners stepping in to provide needed support—from training to new refrigerators, and governments stepping in with more of their own budgets dedicated to immunization. While initially focused on purchasing vaccines for eligible countries, the GAVI partners quickly responded to the overwhelming demand for funds to help repair fragile and faltering immunization and health systems in developing countries. So in addition to supporting countries to introduce new and underused vaccines into their routine systems, a performance-based grant system, called Immunization Services Support (ISS), was developed to provide additional resources for countries to use to strengthen their immunization services on the basis of their own national priorities and needs. GAVI also decided to provide countries with support to improve the safety of all immunization injections. Furthermore, in December 2005 the GAVI Alliance Board took the strategic decision to support developing country health systems, called Health Systems Strengthening (HSS) support, with emphasis on targeting the barriers that hinder efforts to improve immunization. The long-term financing provided by GAVI gives countries the opportunity to conduct better planning, improve vaccine introduction and adoption, and a more reasonable time horizon to take on the responsibility of vaccine procurement. Initially GAVI made five-year commitments to countries, but found that this was not long enough for countries to identify the additional resources required to maintain their improved programmatic performance. Moving to 10-year commitments has significantly improved the credibility and efficiency (in terms of vaccine supply, vaccine introduction, and vaccine delivery) of GAVI’s funding initiatives. GAVI’s financial structure is designed to develop predictable, long-term financing, while converting the world’s poorest countries’ need for vaccines into effective market demand. With a stable financial platform supported by a robust, diverse, and multiyear donor base, GAVI ensures predictable funding in partner countries. By signaling financial stability and long-term committed financing, it is also possible to spur larger markets, accelerate vaccine development, and promote increased production, availability, and lower prices (Box 2).

l l l

l

l

l

Hepatitis B vaccine in all GAVI-eligible countries Hib vaccine in all GAVI-eligible countries Yellow fever in Africa and the Americas, according to regional recommendations Pneumococcal conjugate vaccine in all GAVI-eligible countries. Vaccine supply is currently limited, but it will increase as new manufacturers begin production Rotavirus vaccine in America and Europe, where it has been shown to protect infants and children in randomized, placebo-controlled efficacy studies. Work is ongoing to determine the efficacy and safety in Africa and Asia of the two available vaccine formulations, with final results to be available by 2009 Measles second dose vaccination support will be provided by the GAVI Alliance if it is included in the country’s comprehensive multiyear plan

ISS: ISS is possibly the first truly performance-based program of its kind. GAVI makes an up-front investment in a country’s immunization services, disbursed as a cash grant over a three-year period. Thereafter, countries are eligible to receive an additional US$20 for each extra child they reach with DTP3 vaccine compared with the previous year’s targets. Countries that continue to raise DTP3 coverage rates will continue to receive ISS funding. HSS: Countries apply for HSS support on the basis of national health strategies/plans. Funding should be used to overcome bottlenecks, including (but not exclusive to) (i) health workforce mobilization, distribution, and motivation; (ii) organization and management of health services (e.g., supervision); and (iii) supply, distribution, and maintenance systems for drugs, equipment, and infrastructure Civil Society Organization (CSO) support: CSOs play a crucial part in immunization and health care. In many countries they deliver 10% to 60% of immunization services. GAVI is therefore providing new funding to strengthen CSOs and to encourage the public sector and civil society to work together to plan and deliver sustainable health care.

COUNTRY PROPOSAL REVIEW PROCESS The GAVI Alliance designed a flexible yet rigorous system that allows GAVI to respond effectively to country needs, while only investing in programs of the highest quality. GAVI’s Independent Review Committee (IRC) of experts—based on a peerreview approach—is the foundation on which this system rests. GAVI’s IRC model has been successful, thanks to three characteristics. (i) The committee relies on a wide range of experts in public health, epidemiology, development, and economics. This means that every proposal and report is tested against a number differing, yet equally important, perspectives. (ii) The committee is based on a system of peer review. Not only are IRC members expert in a variety of fields related to immunization, they also have practical experience working in developing country governments. (iii) The committee is independent. It makes its recommendations in an environment free from political considerations. Furthermore, committee members must sign a confidentiality and conflict of interest statement. Committee members who recently have been involved in

Chapter 10: A Paradigm for International Cooperation: The GAVI Alliance

any capacity in the immunization programs in countries under review are not present during the deliberations of the review committee, and do not participate in the decisions for those countries. The IRC is subdivided into three teams, allowing for specialization in GAVI core program areas. To ensure consistency in all deliberations, some members serve on more than one team. These teams are as follows: the new proposals team is responsible for evaluating all country requests for vaccines, injection safety equipment, and cash support to strengthen immunization service delivery systems. The health systems team was formed in 2006 when GAVI created its new HSS window, as HSS support is used to address bottlenecks in a country’s overall health system, not just its immunization delivery system. The monitoring team reviews all annual progress reports and determines whether GAVI should continue support for an approved program.

Process for Review Proposals are reviewed by the IRC at the GAVI Secretariat in Geneva at set times throughout the year. The IRC reviews the proposals in accordance with the policies laid down by the GAVI Board, following the criteria for eligibility and assessment as expressed in the guidelines prepared by the Secretariat. Each new proposal or annual progress report is prescreened for accuracy and consistency by UNICEF, WHO, and the GAVI Secretariat Country Support team before being submitted to the IRC. Their feedback is provided in written form to the IRC for their use during deliberations on the proposals. IRC members on the relevant team read each proposal/report, and select two to three members to review it in depth. Once each proposal/report has been reviewed in detail, the IRC members responsible for in-depth review provide a presentation on its content to the rest of the team. Final recommendations are made during the team’s final deliberation. The IRC makes one of four possible recommendations for each proposal or annual report submission:

Recommendations Approval The application meets all the criteria and is approved for GAVI support. Approval with Clarification The application lacks specific pieces of data, which must be provided (generally) within a month. The requested data must be received before the application is considered officially approved for GAVI support, but the proposal does not need to be review again by the IRC. Conditional Approval The application does not fulfill specific or significant application requirements. Missing requirements must be provided in a subsequent proposal review round to complement the original application. Conditional approvals will be valid for 12 months. If the conditions are not met within one year of the first submission, resubmission of a new application is required. Resubmission The application is incomplete and a full application should be submitted in a subsequent proposal review round.

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Deciding Factors for Each Recommendation The IRC evaluates new country proposals against different criteria, depending on the type of support requested. For all proposals, the IRC evaluates whether the proposal was developed through an inclusive process with many stakeholders. For new and underused vaccines support (NVS) proposals, the IRC evaluates: epidemiological justification; whether the country’s comprehensive multiyear plan for immunization incorporates activities to introduce the new vaccine(s); whether the multiyear plan provides a thorough analysis of cold-chain and logistics capacity; and the country’s plan for financial sustainability. When evaluating a proposal for ISS, the IRC evaluates whether the proposal was developed through an inclusive process with many stakeholders; if proposed future targets for children to be reached with DTP3 are realistic; how estimated ISS financing is factored into multiyear plan activities and budgets. For injection safety support, the IRC evaluates the country’s needs for safe injection equipment, as indicated by the multiyear plan. For each proposal for HSS the IRC evaluates whether the proposal is aligned with the country’s health system policies, plans, and management structures; if the proposal identifies key gaps that impede delivery of immunization and other health services and whether the proposed solutions will effectively address health systems gaps; whether budgets are robust and well sourced (e.g., are unit prices and other cost assumptions included); how the country will monitor progress and track funding flows; and whether the country can sustain progress once GAVI HSS support phases out. The IRC team for monitoring reviews each Annual Progress Report against a number of criteria, including progress against targets set in the original proposal, quality of programmatic reporting, amount of funding used and budget left unspent, feasibility of reaching targets for future years, partner involvement in implementing/monitoring programs, involvement of the Interagency Coordinating Committee (ICC) in planning/coordinating program activities and reviewing reports. The recommendations of the IRC are communicated to the GAVI Alliance and GAVI Fund Boards for final decision. Once the decisions are made, the GAVI Secretariat is responsible for communicating the decisions to the country ICCs, to partners at the regional and field levels, and to UNICEF Supply Division to trigger procurement of the necessary vaccines and supplies.

GAVI GOVERNANCE AND STRUCTURE GAVI Alliance and Fund Boards The GAVI Alliance Board governs policy development and implementation and monitors and oversees all program areas. The Board includes four renewable members: UNICEF, WHO, the Bill & Melinda Gates Foundation, and the World Bank. In addition, there are 13 rotating seats: four for developing country governments, five for donor country governments, and one each for research and technical health institutes, industrialized country vaccine industry, developing country vaccine industry, and CSOs. The GAVI Fund Board shapes the Alliance financial strategy to support implementation of the GAVI Strategic Plan as developed by the GAVI Alliance Board. In this capacity, the Fund Board monitors GAVI income received from multiple sources, validates budgets, certifies availability of funding, and determines funding sources for programs. In addition, the

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Board monitors investments and asset liabilities to ensure financing is available as needed. The Board also provides strategic guidance and support to the United States–based private fundraising work of the Alliance. In November 2007, the GAVI Alliance and Fund Boards took the strategic decision to merge the two organizations, within the framework of a single private foundation, incorporated in Switzerland. The two existing boards will also merge, bringing their combined expertise to one central decisionmaking authority: the new GAVI Alliance Board.

GAVI Secretariat The GAVI secretariat, based in Geneva and Washington, D.C., coordinates Alliance activities including policy development and support to countries.

GAVI Fund Affiliate Board The GAVI Fund Affiliate was established to enter into pledge agreements with IFFIm donors and assign these pledges to the IFFIm Company for eventual program disbursement. GAVI Fund Affiliate Board reviews and approves program-funding requests, and makes subsequent requests for funding to the IFFIm. GAVI Foundation The GAVI Foundation is a Swiss foundation registered in the Geneva Register of Commerce. The Foundation’s charitable mission involves providing support for GAVI Alliance programs and the GAVI Secretariat in Geneva. The GAVI Foundation Board ensures that the Foundation complies with Swiss law and maintains its charitable status (Box 3).

GAVI IMPACT Working Group The GAVI Working Group is responsible for the implementation of the decisions of the GAVI Board, and comprises technical experts from GAVI partner institutions. In this capacity, the Working Group also oversees the accomplishment of the Alliance work plan, preparing policy recommendations for Board consideration, and ensuring close coordination of partner activities. The Working Group is chaired by the GAVI Executive Secretary.

Task Teams Time-limited task teams are established to tackle specific technical, policy, or strategy matters. For example, teams have been created to advise the Board on its health system–strengthening window, support to civil society, immunization financing, and vaccine supply issues.

Traditionally, most new international aid programs start tentatively by supporting a few targeted countries, and then, if appropriate, expanding to more countries. The GAVI partners decided to break the mold and instead to define a list of eligible countries—the poorest countries as defined by gross national income (GNI) per capita—and allow them all to apply for support right away. This enabled often overlooked countries to participate early, and ensured that all eligible countries could readily access the available support. At the World Health Assembly in May of 2000, GAVI issued the first call for proposals. As of end 2007, GAVI has l

 prevented 2.9 million future deaths;  protected 36.8 million additional children with basic

Regional Working Groups The Regional Working Groups (RWGs) were established by partners with a technical presence at the regional level—in most cases WHO and UNICEF—in response to the need to more quickly identify and address the technical assistance requirements of countries, improve communication and streamline efforts, in support of the GAVI and Vaccine Fund processes.

Approved a cumulative US$3.5 billion in support to 73 countries, with which it has

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vaccines (against diphtheria, tetanus, and pertussis); and  protected 176 million additional children with new and underused vaccines. The breakdown of new and underused vaccine coveragea shows

 158.6 million additional children have been immunized against hepatitis B;

 28.3 million additional children have been immunized against Hib; and

 26.3 million additional children have been immu-

National Interagency Coordinating Committees National ICCs represent the leadership and commitment of the national governments and analogous in their operations to the GAVI Board, that is, enhancing partner roles through coordinated action. The roles and functions of ICCs vary considerably from country to country, depending on size, strength of the government, and the presence of other health system coordinating groups such as sector-wide groups.

International Finance Facility for Immunization Entities International Finance Facility for Immunization Board The International Finance Facility for Immunization Company is a multilateral development institution established as a charity with the Charity Commission for England and Wales. The IFFIm Board oversees each bond issuance and develops funding, liquidity, and other operating strategies to safeguard and maximize the value of IFFIm proceeds.

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nized against yellow fever. In 2000, 40% of GAVI-eligible countries had DTP3 coverage below 60%. By 2006, that number had dropped to 11%. Significant declines are now being seen in the price of DTP-HepB vaccine as demand is generated and new players enter the market. Declining prices are vital to ensuring that vaccination programs are sustainable. In 2006, almost 30% of all the vaccine doses purchased by UNICEF for the GAVI Alliance came from developing country manufacturers. Spending on vaccines in the poorest countries supported by GAVI more than doubled from US$2.50 to over US$5.00 per child between 2000 and 2005.

Not all children received all three new and underused vaccines. Therefore, the total figure of 176 million children is not the sum of children vaccinated against hepatitis B, Hib, and yellow fever.

Chapter 10: A Paradigm for International Cooperation: The GAVI Alliance

RESEARCH AND DEVELOPMENT IN THE CONTEXT OF THE GAVI ALLIANCE The global vaccine research community includes many diverse players, each contributing in different ways with vastly heterogeneous resources and distinct agendas. Much can be gained by coordinating agendas and goals to render efforts complementary, avoid duplication, and maximize the use of limited resources. The many partners involved in vaccine research and development activities and epidemiological studies in both industrialized and developing countries are represented in the Alliance in a truly synergistic effort. Partners include governmental research institutes, academic research programs, large vaccine manufacturers, biotechnology companies, units within Ministries of Health, etc. There are also partners that are not primarily engaged in research but who, as implementers of immunization, will provide critical input to researchers to advise them on what is needed, feasible, and desired at the front lines of primary care, and in contrast, what cannot be readily incorporated into primary care regimens. With all of the current activity in vaccine R&D, it is important that in the context of GAVI, efforts focus on identifying those gaps where an alliance can have a strategic advantage. Thus, while the GAVI partners recognize that a high priority lies in HIV/AIDS and malaria, given the massive global effort to these projects worldwide, the Alliance decided to prioritize other vaccines that are receiving less attention. In addition, even if these vaccines become available, many of the poorest countries lack the infrastructure to put these vaccines into public health use efficiently. Therefore, initially, GAVI partners decided to initially place their R&D focus on vaccines that have a lower technical risk and a greater potential for more near-term development and introduction. To identify these priority vaccines, members of the GAVI Task Force on Research and Development (R&D TF) conducted wide consultation with the R&D community to develop consensus. The criteria for choosing the disease-specific projects were based on a number of considerations. l

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Either no currently registered vaccine or for which the existing vaccines have notable drawbacks that severely limit their public health usefulness (e.g., the existing vaccines are not immunogenic in infants yet that age group is an epidemiological target for vaccination) High potential impact in terms of disease mortality rate and disability-adjusted life years (DALYs) Nonavailability of alternative solutions to managing the disease Good potential for changing/improving the immunization system for the future in terms of capacity building and promoting behavioral or system change High degree of feasibility with available tools and infrastructure; political commitment

The consultation process led to a consensus that the three vaccines that should receive high priority in the context of GAVI are: Streptococcus pneumoniae, rotavirus, and Neisseria meningitidis group A (which may be approached either as a monovalent group A, a bivalent group A/C, or a quadravalent group A/C/Y/W135 vaccine). To accelerate the availability of these vaccines, in June 2002 the GAVI Board decided to fund special projects, called accelerated development and introduction plans (ADIPs), to focus on rotavirus and pneumococcal vaccines. The ADIPs

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define critical actions to establish the value of the vaccine, to communicate this value of to the key decision leaders, and to deliver the value by ensuring supply and delivery systems are in place. A meningococcal ADIP was not created at that time because funding had been already provided directly by the Gates Foundation to establish the Meningitis Vaccine Project. In November 2006, the GAVI Alliance Board made the strategic decision to support countries to introduce rotavirus and pneumococcal vaccines. It will soon take a decision on which other new vaccines it will support in the future.

LOOKING TO THE FUTURE: ISSUES AND CHALLENGES Vaccine Supply The relatively small size (in revenues) of the developing country vaccine market has resulted in reduced private sector investment in relevant vaccines and thus reduced production capacity. GAVI, with private sector involvement and a longterm perspective, lends a much needed stability to delivery systems, demand creation, and vaccine supply. GAVI has already demonstrated that if the public sector can work to help make the developing country vaccine market more attractive to vaccine manufacturers, children living in the poorest countries will live healthier lives by having access to better and more effective vaccines.

Sustainability Financial sustainability is crucial to the success of immunization programs in countries and will be the measure of GAVI’s success in the long term. However, the challenge of creating systems that are sustainable beyond the initial time of investment is one of the most critical issues facing all areas of development—not just GAVI. To clarify the aim of strategies developed to enhance the sustainability of programs, the GAVI Board adopted a new definition of financial sustainability: ‘‘Although self-sufficiency is the ultimate goal, in the nearer term sustainable financing is the ability of a country to mobilize and efficiently use domestic and supplementary external resources on a reliable basis to achieve target levels of immunization performance.’’ In this way GAVI partners recognize that for the foreseeable future, maintaining high quality immunization programs in the health systems of the poorest countries will require continued external support—from donor governments, NGOs, the private sector, and individuals, support that has flagged in too many countries in recent years. Cofinancing, introduced by GAVI in 2007, means countries share the cost of the vaccines supplied by the GAVI Alliance. The intention is to ensure that immunization programs are sustainable in the long term. GAVI-eligible countries have been grouped according to their expected ability to pay, and the cofinancing levels vary across the different groups. GAVI will conduct an evaluation of the cofinancing policy in 2009. On the basis of the outcomes of the evaluation, current cofinancing levels, country groupings, and eligibility criteria are expected to be revised in 2010.

Safety and Waste Management Worldwide, each year, the overuse of injections and unsafe injection practices combine to cause an estimated 22.5 million hepatitis B virus infections, 2.7 million hepatitis C virus

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infections, and 98,000 HIV infections. Although injections given as a part of immunization programs account for a very limited proportion (approximately 5%) of the injections delivered and are widely considered the safest of all delivered, there is a growing body of data demonstrating the safety of immunization programs throughout the world need to be improved. On the basis of the principle of ‘‘do no harm,’’ the GAVI partners acknowledge the importance of improving the safety of immunization programs and have focused special attention on safety in relation to the other elements of immunization programs. Appropriate disposal of medical waste is an important element of efforts to improve the safety of national immunization programs and should be based on the principle that the ‘‘polluter pays.’’ Although at present there are very limited environmentally sound options for safely eliminating waste, the Alliance is committed to supporting countries in their immediate action, using the best practices available to minimize the risk of exposure to medical wastes for staff and the community. GAVI partners encourage further investment in the development of new environmentally sound, reasonably priced methods for disposing of medical wastes.

GAVI Fund: Future Needs The GAVI Fund was created to provide resources to countries to fill critical gaps in funding for immunization services. The GAVI Alliance Board identified that the most critical gaps at present are the weak health service infrastructure in many countries, the delay in introducing new life-saving vaccines as they become available, and the need to improve safety of immunization programs.

As the country programs progress, new gaps are likely to become apparent, and new vaccines now on the horizon will need to be introduced into routine immunization systems. Therefore the GAVI Fund will need to continually attract new resources.

Maintaining a Robust Learning Curve It is important to retain the perspective of GAVI as an experiment. By joining together in this public-private alliance, the GAVI partners have committed to working together in new ways. As new hurdles arise, the Alliance has been able to reexamine and revise its policies and directions. For example, GAVI Alliance expanded its early focus on vaccines to include support for health system infrastructure. Much of the early work of the Alliance has been dedicated to the development of policies and the proposal process for the GAVI Fund, and providing the eligible countries the technical support they need to apply for support. Now that virtually all eligible countries have been approved for support, the GAVI partners are shifting emphasis to supporting implementation of improved immunization programs and issues of sustainability. Looking ahead, GAVI partners will need to continually monitor their impact to assess whether their efforts, and support from the GAVI Fund, have in fact helped countries meet their targets. The role of the partners—most of which have worked in immunization long before GAVI came together—cannot be stressed enough. With so much attention focused on the GAVI Alliance as a new contributor to the global immunization community, it must be remembered that all of the progress logged over the past years has resulted from partner commitments—staff, resources, and financial support—in countries, regions, and at the global levels.

11 Economic Analyses of Vaccines and Vaccination Programs Benjamin Schwartz National Vaccine Program Office, U.S. Department of Health and Human Services, Washington, D.C., U.S.A.

Mark L. Messonier National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

INTRODUCTION Economic analysis of new vaccines has become increasingly important, contributing to decisions ranging from vaccine development to vaccine recommendation and program implementation. Over the past decades, the cost of developing and licensing pharmaceutical products has increased markedly. An estimate based on the cost of new drug development suggests costs ranging from $400 to $800 million in 2000 dollars. This estimate is nearly four times higher than costs estimated previously using a similar methodology in 1987 dollars (1). Costs associated with development of vaccines that use new technologies or contain novel adjuvants may be even higher. As vaccine development costs have risen, so too have vaccine prices. Driven by the high prices of new vaccines, the cost of fully vaccinating a child in the United States in 2008 was more than $1200 in the private sector and $850 in the public sector (2). This cost exceeds by more than twofold the cost to fully vaccinate a U.S. child in 2001 (3). Adolescent vaccination adds about $500 to these costs, driven by the three-dose human papillomavirus (HPV) vaccine, which costs $120 per dose. This increase has outstripped expansion of appropriations to support state-based vaccination programs, forcing some states to decide which vaccinations can and which cannot be provided for children who need assistance but do not qualify for coverage under the Vaccines for Children program. Recognizing the challenges to the U.S. vaccine-financing system and the value of stimulating continued development of new products, in 2004 the U.S. National Academy of Science’s Institute of Medicine (IOM) recommended major changes to increase incentives for new product development, increase stability of vaccine supply, and enhance coverage. One recommendation calls for insurance mandates and subsidies for current and future vaccines based on the values of the products to society using a methodology that considers ‘‘such factors as reduced health expenditures, enhanced quality of life, and increased labor productivity’’ (4). Although the IOM recommendations have not been implemented, the suggestion that vaccine prices, in part, should reflect their societal value raises some essential questions: can an economic analysis of a new vaccine appropriately determine its societal value? Are the analytic strategies sufficient? Can the necessary data be obtained? And would the results be viewed by manufacturers and payers as valid?

Economic analysis of new vaccines is also becoming an important tool in developing recommendations for their use. In the industrialized world, as infectious disease mortality has declined, new vaccines generally have a much greater impact on morbidity than on death. Consequently, recommendation of a new vaccine or expansion of existing recommendations may be justified largely by the prevention of morbidity and its associated costs. The 1995 U.S. recommendation for varicella vaccination was supported by an economic analysis that indicated savings of $5.40 for each dollar spent on the vaccination, considering both direct and indirect costs. However, when only direct costs are considered, vaccination represents a net cost as the ratio of indirect to direct cost savings is almost 5:1 (5). Thus, the ability to appropriately characterize the full range of societal costs and benefits associated with vaccination and to interpret these results as they apply to policy decisions represents another challenge to economic analysis. In developing countries, infectious diseases still account for a majority of childhood deaths and a substantial portion of the overall population burden of disease. Introduction of new vaccines against hepatitis B, Haemophilus influenzae type b, pneumococcal infections, and rotavirus has been slow, in large part because of their costs. Increased funding from international donors, coordination through the Global Alliance on Vaccines and Immunization, and the development of combination vaccines that include antigens appropriate for a developing country program have accelerated the pace of new vaccine introduction. Improved planning and coordination among developing countries, donor organizations, and industry have occurred with the formation of vaccine-specific groups coordinating advanced development and introduction plans (ADIPs). ADIPs facilitate new vaccine introduction by documenting for decision makers the burden of disease and the value of prevention, with the objective of obtaining an advanced commitment to introduce a new vaccine at a target price. This commitment allows industry to develop production capacity for a predictable demand and to provide deep discounts in price that will allow the vaccine to be introduced in a country with limited resources (6). While coordination and external financial support have been critical to make new vaccine introduction feasible, economic analysis remains important to decision-making as at some point countries may transition from donor to domestic funding.

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As economic analysis assumes greater influence on decisions for vaccine development and pricing, introduction and recommendation, and program financing, it is important to understand the effect of choices in analytic model, perspective, and parameters on the outcome of the analysis. The purpose of this chapter is to describe the types of economic analysis commonly used, their components, and the advantages, disadvantages, and controversies that exist with each approach. In addition, some of the limitations and inconsistencies of vaccine economic analyses published in the medical literature will be highlighted, emphasizing the importance of critically assessing this body of work.

TYPES OF ECONOMIC ANALYSES Cost-Benefit Analyses Three types of analyses are commonly used to assess the economic rationale for immunization or other health-related programs: cost-benefit (CB), cost-effectiveness, and cost-utility analyses (Table 1) (4). Cost-benefit analyses (CBAs) compare the monetary benefits from implementation of a health program with its monetary costs. All values in a CBA, including health outcomes, are converted to monetary units, resulting in an outcome that is expressed as money saved or spent. The results of a CBA are typically presented either as the net present value (NPV) of the program or the CB ratio. NPV is defined as the difference between the discounted benefits and discounted costs of the intervention, or NPV ¼

PN


t ðB  CÞt , where
¼ 1=ð1 þ rÞ , r, the discount rate as a decimal, and t, number of years analyzed (between t ¼ 0 and N). Alternately, results of the analysis can be expressed by the CB ratio, which is the ratio between the discounted costs and benefits of the intervention, or t

t¼0

t

N P

CB ¼

t¼0 N P t¼0


t Ct :
t B

results in the ethical dilemma of valuing life differently by countries or by genders or ethnic groups within a country (8). While alternate methods have been proposed to estimate the economic value of a life, no method is accepted universally. It is also difficult to assign a monetary value to the pain and suffering that occur with an illness. The contingent valuation method (CVM) has become a common strategy to define these values. Studies that use the CVM to estimate the value of a health program are commonly called willingness-to-pay (WTP) studies. A common approach used in contingent valuation studies involves surveying a population for the maximum they would spend to avert or decrease the risk of a specific hypothetical outcome (9). Drawbacks with this approach include results being sensitive to how the outcome is presented; differences in valuation between those with different socioeconomic status, levels of education, or experience with a disease state; and difficulties setting risk or probability values for very rare events such as adverse events following vaccination. An alternative method taken, in part, from product development and marketing is conjoint analysis. Using this method, preferences are expressed for programs or products that have a range of defined attributes jointly as a ‘‘product profile’’ (10). Although this approach has not been used extensively for analyses of vaccines or vaccination programs, it appears to be a useful strategy to better value a product or program. Because of the use of surveys to define values, the CVM and conjoint analyses are called stated-preference methods.

t

For new vaccines, where the price per dose has not yet been set, a ‘‘break-even’’ price can be calculated where the net costs and benefits are equal (NPV ¼ 0). Because all values and outputs are expressed in monetary terms, a CBA is useful to compare the economic value to society of programs with different impacts, such as a health program with another (nonhealth) type of program. Another advantage of this approach is that the results are expressed in a way that is easily understood by policymakers and the public. An important shortcoming of CBA is the need to quantify all program impacts in monetary terms. The ‘‘human capital’’ approach assigns a monetary value to disability or death on the basis of the NPV of the expected future earnings lost (7). This

Cost-Effectiveness Analyses Cost-effectiveness analysis (CEA) assesses the value of a program by calculating the expenditure per health outcome achieved (net cost/net health effect). Common outcomes include cost per case prevented and cost per death averted. Average cost-effectiveness (ACE) is used to describe costs and outcomes for an independent program, ACE ¼ CA / EA, where A is a program or strategy. Incremental cost-effectiveness (ICE) describes the ratio between costs and outcomes for programs that are being compared, ICE ¼ CA  CB / EA  EB, where A and B are competing programs or strategies. Finally, marginal cost-effectiveness (MCE) describes the costs and outcomes within a single program to show cost of expanding, MCE ¼ CAI  CA / EAI  EA, where AI is an extension of A. Because the denominator is a specific health event, CEA is used to choose between alternative interventions that are aimed at achieving a similar outcome. For example, CEA has been used to determine whether cholera vaccination in conjunction with rehydration therapy in refugee settings is cost effective in preventing morbidity and mortality compared with rehydration therapy alone (11). An advantage of CEA compared with CBA is that, by comparing similar outcomes, it is unnecessary to convert health outcomes into monetary values.

Table 1 Comparison of Characteristics of Cost-Benefit, Cost-Effectiveness, and Cost-Utility Analyses Cost-benefit analyses

Cost-effectiveness analyses

Cost-utility analyses

Outcome measure

Net present value

Calculation of outcome measure Useful for comparing

Benefits-Costs

Cost per health outcome averted (or gained) Net costs/Net health effects

Cost per health utility (e.g., QALY) gained Net costs/Health utilities gained

Health program A vs. health program B (with same health outcome)

Health program A vs. health program B same (with different health outcome)

Health programs vs. nonhealth programs

Chapter 11: Economic Analyses of Vaccines and Vaccination Programs

A limitation of this approach, however, is that CEA can be used only if the interventions being compared share a common outcome measure. For many new vaccines in the United States where the impact on morbidity far outweighs that on mortality, CEA is of little usefulness in comparing value (e.g., between prevention of varicella, rotavirus gastroenteritis, and pneumococcal otitis media). In addition, the results of CEA are more difficult to apply in making policy. For example, an analysis of infant pneumococcal conjugate vaccination in the United States suggested that at a cost of $58 per dose, vaccination would cost society $160 per episode of otitis media prevented and $3200 per episode of pneumonia prevented (12). Although one could assess whether these costs are reasonable by directly comparing them with the costs of treatment, such an approach would ignore the benefit of preventing the suffering associated with an illness.

Cost-Utility Analyses Cost-utility analysis (CUA) is a subset of CEA. In CUA, as with CEA, outcomes are expressed as cost per change in health status; thus, health outcomes do not need to be converted into monetary equivalents. However, a common metric is established that allows comparison of qualitatively different health outcomes. Quality-adjusted life years (QALYs) and disabilityadjusted life years (DALYs) are examples of widely used utilities (7,8,13–15). The calculation of cost and impact in CUA is identical to that for CEA except that the denominator is expressed as health utilities gained. The CUA is most useful in three settings: (i) when comparing health programs that have different disease impacts, (ii) when summarizing the overall impact of a program that affects both morbidity and mortality outcomes, and (iii) when comparing a program that primarily affects mortality with one that primarily affects morbidity. Because results of CUA are expressed as cost per health benefit, this method cannot be used to compare a health program with a nonhealth program. QALYs have become the most common utility used in CUA and provide a method to compare health outcomes where both the quality and duration of life are affected by a disease. QALYs represent the sum of each year of life multiplied by the quality of each of these years, expressed on a scale ranging from 1 (perfect health) to 0 (death). Several approaches have been used to establish quality values for morbid conditions. Using the standard gamble approach, respondents are asked to state a preference between living with a disability and an alternative that includes a risk of death (p) or of perfect health (1  p). The probability at which the respondent considers the two options equivalent represents the health utility. With the time trade-off method, respondents are asked how much perfect life they would trade to avoid life with a defined health condition (6). Although recommended methods to derive health utilities have been published by the U.S. Public Health Service Panel on CostEffectiveness in Health and Medicine (16), several utility scales are available, and this remains an area of investigation. Although CUA has been used more frequently in recent years to evaluate and compare health programs, there are potential concerns with this method. Most importantly, the results of a CUA depend on the utilities assigned to acute and chronic morbid states. Preferences expressed using standard gamble or time trade-off methods are affected by the description of the health condition, the questions asked, and the characteristics of the respondents. Utilities for morbidities

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prevented by childhood vaccination may also depend on who is interviewed as proxy respondents: parents, in general, or those whose children experienced the relevant disease state, or a random sample from the community. In a study of preferences for health states prevented by pneumococcal conjugagte vaccination, Prosser et al. found significant differences in time trade-off amounts between parents and a community sample for simple and complex otitis media and for moderate pneumonia, with the community sample placing a higher value on preventing these outcomes (9). For policymakers, understanding the meaning of a health utility is more difficult than evaluating a positive or negative value in a CBA or a cost per health outcome in a CEA. Various standards have been proposed to determine whether a cost per health utility is a ‘‘good buy.’’ For developing countries, WHO defines a cost per DALY averted of less than three times the per capita gross domestic product as cost effective and ratios below the gross domestic product per head as highly cost effective (17). Another approach is to compare the cost-effectiveness of a new vaccine recommendation with that of other recommended preventive services, although comparison is hindered by different methods used to define cost-effectiveness of different interventions (18,19).

Examples of Economic Analysis Used to Evaluate Vaccines and Vaccination Programs Each type of economic analysis has been used in assessing vaccines and vaccination programs. CUA has been used to compare and determine priorities among vaccinations and other clinical preventive services because of the flexibility of this approach to compare health programs with different mortality and morbidity outcomes (19). CUA results were also used by the IOM to define vaccine development priorities in categories based on the cost per QALY saved (20). CUA, CEA, and CBA have all been employed to assess the impact of a new vaccine or a change in the vaccination schedule. In a summary of 15 economic evaluations of pneumococcal conjugate vaccination published between 2002 and 2006, twelve evaluated cost-effectiveness, eight expressed cost-utility outcomes, and one reported CB ratios (21). Programmatic issues, such as the use of reminder-recall or other interventions to increase vaccination coverage, have been assessed by CEA, with the outcome measured as cost per person vaccinated (22). Stated-preference methods have been used to define values for safer or combination vaccines (23,24).

COMPONENTS OF ECONOMIC ANALYSES The outcomes of a vaccine economic analysis depend on the choices of the investigator in the type of model used, and the impact and cost parameters included and their values. Although the U.S. Public Health Service Panel on CostEffectiveness in Health and Medicine recommended a standard methodology to facilitate comparison across different studies and types of interventions (16,25–27), articles published in the medical literature use a range of approaches (28), making careful evaluation of the methods and parameter values particularly important.

Developing the Model The evaluation of a vaccine or vaccination program using CBA, CEA, or CUA begins with creating a conceptual model (29). A

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Figure 1 Example of a decision tree model for a hypothetical vaccine.

decision tree model describes chance events and decisions over time, with the range of possible outcomes and the probability of their occurrence represented graphically (Fig. 1). Typically, a decision tree begins with a ‘‘decision node’’ (presented as a box), where the alternative interventions are distinguished from one another (e.g., implement or not implement an immunization program). The next levels of the decision tree describe the consequence of the previous decision and its probability. Examples include vaccinated/not vaccinated, adverse event/ no adverse event, and disease/no disease. The probability of each of these events is represented by a ‘‘chance node’’ (presented as a circle). Chance nodes may have several different possible outcomes. However, the alternative outcomes of a chance node must be mutually exclusive and exhaustive, and the sum of the probabilities of a chance node will always equal 1. The final level of the decision tree, called the ‘‘terminal node’’ (presented as a triangle), represents the ultimate health outcome of the sequence of events along a branch of the decision tree. Different adverse events and types of disease should be specified separately along with the consequences of each (e.g., no sequelae, sequelae, or death). Many of the computer software programs for conducting economic analyses begin with the creation of a decision tree. However, this graphical representation is not required, and some investigators prefer to use spreadsheet software. State-transition models are more efficient in evaluating policy options when a series of health events occur over time. A Markov model is one type of state-transition model and includes a set of mutually exclusive health states, characterizing transi-

tions that occur between states over time in populations defined by characteristics such as age and gender. For example, a model assessing HPV vaccine would include persons with HPV infection, various grades of histological lesions, and local or more widespread cancer. Analysis is done using a cohort simulation where a hypothetical group of persons is tracked simultaneously through the model or using a Monte Carlo simulation where individuals move through the model individually, with characteristics and transition probabilities drawn randomly on the basis of distributions derived from the data (30). Dynamic models may be optimal when herd immunity effects occur such that the rate of infection among susceptibles depends on the number of infectious people in the population and the chance of effective contact between them (31).

Estimating Impacts One of the greatest challenges to economic analysis of a new vaccine is determining its impacts, defined by the incidence of potentially preventable conditions and the vaccine efficacy for each. For a new vaccine, efficacy is initially defined in randomized, controlled pre-licensure trials but, as few such trials are done, results are often applied to other populations and other vaccination schedules than those studied. Differences in population characteristics, disease incidence, and, where relevant, the serotype distribution must all be considered in the analysis (Table 2). Among the 15 economic analyses of pneumococcal conjugate vaccine (PCV) in industrialized countries reviewed by Beutels et al. (21), vaccine efficacy estimates were all derived

Chapter 11: Economic Analyses of Vaccines and Vaccination Programs

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Table 2 Uncertainties in Defining Disease Impacts of a New Vaccine

Table 3 Examples of Direct and Indirect Costs Often Considered in Economic Analyses of an Immunization Program

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Differences between the pre-licensure study population and the population considered in the economic analysis Differences in disease incidence between geographic areas or by season or year Differences in strain or serotype distribution Uncertainty around point estimates, especially for less common outcomes Indirect (herd immunity) effects Impacts of vaccination on distribution of strains (serotypes) Impacts of vaccination on antimicrobial resistance (treatment outcomes)

from clinical trials in the United States and in Finland. Most analyses used local data on the distribution of pneumococcal serotypes and disease incidence. However, it is unclear whether the substantial differences between analyses in incidence reflect true variation or differences in the sensitivity of surveillance based on diagnostic practices or the sensitivity of etiological diagnosis. For the developing world, an analysis of PCV cost-effectiveness extrapolated vaccine effectiveness results from The Gambia (32) to 72 countries, adjusting effectiveness against all-cause mortality based on under-five-year-old mortality rate categories but not taking into account differences in serotype distribution (33). Herd effects (e.g., reduction in disease burden among unvaccinated persons resulting from reduced exposure and acquisition of infection in a community), which generally cannot be measured in pre-licensure trials, can have a large positive effect on the impact of a vaccination program and thus, results of economic analyses. Conversely, changes in serotype distribution with replacement of vaccine types by those not included in a multivalent vaccine would have a negative effect on the overall impact and economic value of vaccination. An analysis of the cost-effectiveness of PCV in the United States that did not incorporate herd effects estimated prevention of 38,000 cases of invasive pneumococcal disease during a fiveyear period, whereas including herd effects increased the number of cases averted to 109,000. The costs per life year saved were $112,000 in the former and $7500 in the latter analyses (34). Characterizing vaccination program impacts is especially problematic for influenza where disease burden and vaccine efficacy vary markedly from year to year depending on the viruses that circulate and the antigenic match with the strains included in the vaccine. Longitudinal estimates of influenza mortality and hospitalization in the United States are available (35,36), but data on vaccine effectiveness are more limited. Limited use and accuracy of etiological diagnosis, the contribution of influenza to morbidity and mortality from secondary bacterial infections or cardiorespiratory disease, and age-specific differences in vaccine effectiveness all complicate economic analyses for influenza vaccination.

Estimating Costs Direct and indirect costs are typically considered in economic analyses of vaccination programs (Table 3). Direct costs include those related to health events that would occur with and without the program, and the costs of the vaccination program itself. The direct costs of health events include costs of medical

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l

Vaccination program costs – – – – –

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vaccine vaccine administration medical care visit(s) for vaccination medical care visit(s) for adverse events (inpatient/outpatient) medication for therapy of adverse events

Transportation to and from medical services Child care Housekeeping Vaccine research and development

Indirect costs l

Change in productivity from illness or vaccine adverse event – time lost from death and illness – time lost for medical care visits – decreased productivity while at work due to illness or sequelae – time lost while caring for an ill family member

l l

Forgone leisure time while ill or caring for an ill family member Intangible costs (e.g., pain and suffering)

Specific costs included will depend on the type of economic analysis and the perspective taken.

care visits, diagnosis, and acute and long-term treatment. The costs of transportation to the doctor’s office or hospital and of housekeeping or child care services during a period of illness are also direct costs. The direct costs of the vaccination program include the cost of vaccine, administration, and the medical care visit including travel costs. When a new vaccine is given on the same schedule as the one that is currently recommended, costs of the vaccination visit may be omitted because the new vaccine would be administered at the same visit; thus, there would be no incremental costs (37). Program costs such as the costs of storing and transporting vaccine, maintaining the cold chain, and training health care workers to administer a new vaccine are often not included in analyses for industrialized countries but may be important for vaccines administered through the public sector in developing countries, where the incremental costs are greater. Treatment of adverse events following immunization must be added to costs, although for most vaccines, the magnitude of these costs will be small relative to other components of the model. Other costs may be considered in certain settings, depending on the purpose of the analysis. Vaccine development costs may be relevant if the analysis focuses on priorities for developing new vaccines. For a new vaccine to be used in developing countries, it may be reasonable to consider foreign exchange costs, discounting costs borne in local currency. Among indirect costs, the greatest impact typically comes from what are described as losses in productivity due to time lost from work, disability, or premature death occurring as a

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consequence of illness. Some analyses also include the costs of pain and suffering from illness, adverse events, or an additional injection as indirect costs, with data derived using methods such as WTP. For many recently recommended vaccines in the United States, inclusion of indirect costs has had a substantial impact on the results of the analysis; this impact can be highlighted by comparing the value of a new vaccine from the health care payer’s perspective—where only direct costs are considered—with the societal perspective where both direct and indirect costs are included. For example, one study estimated the breakeven perdose rotavirus vaccine cost in the United States at $12 from the health care payer’s perspective compared with $42 from the societal perspective (38). Routine varicella vaccination of U.S. children has been estimated to save $5 for every dollar invested from a societal perspective but, from the payer’s perspective, would cost about $2 for each case of chicken pox prevented (5). Although indirect costs are routinely included in economic analyses and are recommended to be used by the Panel on Cost-Effectiveness in Health and Medicine (7), there is no solid evidence supporting the assumption that, in the absence of illness, national productivity would increase by the magnitude estimated. If workers use vacation or sick leave days when ill or when caring for a sick child, if they trade work shifts with a coworker when they make a medical care visit, or if industries would cut their workforce rather than increase output if worker absences were eliminated, then it would be incorrect to link societal productivity with illness prevention. Some economists respond by equating this value with lost leisure time, suggesting that lost leisure when ill or caring for a sick child has the same societal value. In developing countries where there is a large unemployed labor pool, work loss from illness or disability or the need to care for an ill family member would likely have minimal impact on overall productivity. While a ‘‘friction cost’’ approach has been proposed as an alternative to the ‘‘human capital’’ approach to estimating indirect costs (39), its use is controversial (40). Once decisions have been made regarding what costs to include in an economic analysis, the next step is to assign monetary values. For medical care and treatment, the use of costs, rather than price or charge, is recommended because the cost more closely reflects the true value placed on the service. Several databases, including Medicare payments or the payments made by large managed care organizations, are available to estimate costs of medical care. Charge data can be converted to costs by using published cost to charge ratios (7). Indirect costs have generally been calculated from the number of days lost due to illness multiplied by the value of a workday. Some studies compute the indirect cost of lost labor taking into account age- and gender-specific participation in the workforce and wages (41), available for the United States from the Bureau of Labor Statistics (42,43). Work loss estimates are adjusted for weekends and holidays. In general, women are assumed to care for sick children. Whereas race and ethnicity and socioeconomic status are associated with incidence for some illnesses, we are not aware of studies that consider these factors in calculating indirect costs. Because indirect costs of recently licensed vaccines have outweighed direct costs and because methods for defining indirect costs vary, public health personnel and policymakers must scrutinize the methods in economic evaluations and understand the choices made and estimates used. In four recent analyses of influenza vaccine (44–47), the number of work loss days estimated for an ill adult ranged from 0.52 to 2, and the value of a workday ranged from $93.40 to $120. One study (44) estimated

the cost of influenza vaccine and its administration at $4 on the basis of data published five years previously, while a second study published a year later from the same group used an estimate of $10 (45). A 1998 study of the cost-effectiveness of infant rotavirus vaccination assumed 3.4 workdays lost whether medical care was needed or not (38), whereas a more recent analysis by many of the same authors more conservatively estimated 1 workday lost for a case requiring only home care (37). Varying estimates of the value of human life may also affect the results. The human capital approach values life in a manner similar to capital equipment where productive output is lost or diminished because of premature death or disability, the value of the death being equivalent to the NPV of expected future earnings. This approach has been criticized as undervaluing persons who are older or not in the workforce. Also, because the value of life will differ between countries, international comparisons of health states would tend to favor conditions that affect the populations of industrialized countries. Values obtained using the WTP approach tend to be higher than that obtained using the human capital approach and may vary considerably between studies.

Discounting Costs and Health Effects Quite often the costs and benefits of a vaccination program accrue at some point in the future, long after the vaccination itself. Because society values accruing benefits early and delaying costs until later, a method called discounting is used to adjust all costs and benefits to their present value. It is important to note that discounting is not an attempt to adjust for inflation, because even with no inflation, individuals will prefer benefits that occur now compared with that some time in the future. Although no single discount rate is used universally, the Panel on Cost-Effectiveness in Health and Medicine recommends using a rate of 3% (16). Other rates may be chosen to facilitate comparison with other studies and in sensitivity analyses. The importance of discounting will vary for a vaccine where the impact is acute and long-term consequences of infection are rare (e.g., influenza) and one where the health impacts occur long after the vaccine was administered (e.g., hepatitis B, HPV, herpes zoster).

Distribution of Costs and Benefits Economic analyses generally assess costs and benefits using a health care payer or a societal perspective. The health care payer perspective includes only direct costs, whereas a societal perspective considers both direct and indirect costs. Routinely recommended vaccines in the United States such as PCV, varicella vaccine, and rotavirus vaccine are cost neutral or cost saving to society at current vaccine prices but represent a net cost to health care payers who do not benefit from the productivity cost savings accruing to society. Even when only direct costs are considered, the health care organization that bears the costs of an immunization may be different from the one that benefits. For example, because of the high annual turnover in the client population of a managed care organization, the costs of a prevention program for hepatitis B or herpes zoster among older adults may be borne by one organization, while another reaps the benefits of disease prevented for years in the future. In many developing countries, the Ministry of Health bears the majority of the burden for the direct costs of a vaccination program, while the benefits of direct costs averted are distributed throughout the population.

Chapter 11: Economic Analyses of Vaccines and Vaccination Programs

SENSITIVITY ANALYSES Parameter estimates in economic analyses are inherently uncertain; in part because data on disease and its prevention are limited by the populations studied and because future events (e.g., occurrence of herd immunity or emergence of replacement serotypes) cannot be known. In economic models, sensitivity analyses are important to assess the uncertainties that surround the estimates of cost and benefits, and the costeffectiveness ratios they produce. In a sensitivity analysis, the investigators typically define the range of estimates for potential parameters in the model on the basis of statistical uncertainty in the clinical studies from which the parameters are taken or on expert opinion. The impact of changing each parameter between plausible extremes can be determined one by one in univariate sensitivity analyses. This type of analysis is useful in identifying the parameter(s) to which the overall result is most sensitive. However, a univariate sensitivity analysis cannot determine the true range in possible outcomes or a confidence interval around the baseline result. Probabilistic sensitivity analysis using Monte Carlo simulation randomly varies the parameters across the reasonable range according to an assumed probability distribution and can approximate a confidence interval for a cost-effectiveness ratio (48). This approach also allows one to graphically represent the range of possible outcomes and define the proportion that meets a defined standard for cost-effectiveness (31).

Table 4 Key Items to Include in a Published Report of an Economic Analysis 1.

Approach l l l

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2.

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l

l

l l l l

l

The results of economic analyses can have a substantial influence on policy decisions such as the recommendation for routine use of a new vaccine. Because subtle biases can be introduced into an analysis based on choices investigators make in developing the cost-effectiveness model and defining parameters, careful evaluation of the methods and results are critical. Guidance on methods for conducting (16,25–27), reporting (49), and presenting economic analyses to policymakers (50) have been proposed. Key elements that should be included in an economic analysis are shown in Table 4. Special care should be taken in evaluating the parameters to which the model is most sensitive. For example, when considering disease burden, what data are available and are they generalizable to the population being considered? If data from multiple studies were available, how were they summarized and how was a base-case estimate selected? For a disease that results in significant morbidity but in little mortality, what data are used to determine work loss among adult caretakers of sick children and what economic values are assigned to their productivity? On the basis of the results of the sensitivity analysis, can the results of the study be considered robust across the range of plausible inputs or should the analysis be deemed noncontributory to decision-making? Finally, what additional studies or data would be useful in refining the analysis? The science of economic analysis has progressed markedly in the past decade as new methods have been applied and standard approaches recommended that, if implemented, will improve the quality and comparability of studies. At the same time, inherent limitations in the data available at the time a new vaccine is licensed and evolving methodological issues will limit the ability to conduct an economic analysis so that, as the U.S. IOM has suggested, the results could be used to establish the value of the product. Experience with recently licensed

3.

Identification of outcomes of interest Definition of outcome measures Description of model Modeling assumptions Diagram of event pathway or model Software used Sources for and data on model parameters (e.g., disease burden, program effectiveness, cost data) Strategy for summarizing data to develop base-case estimates when multiple data points are available Statement of methods for obtaining expert opinion where it is used to estimate parameters Method to define preferences and preference weights Critique of data quality Statement of year of costs Statement of method used to adjust costs for inflation Statement of type of currency Statement of discount rate

Results l l l l l l

4.

General design of the analysis Target population for intervention Program description (e.g., case setting, model of delivery, timing of intervention) Description of comparison program Time horizon Perspective of the analysis

Data and methods

l

INTERPRETING AND COMPARING ECONOMIC ANALYSES

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Results of model validation Aggregate cost and impact of the program Results of base-case analysis Results of sensitivity analysis Statistical estimates describing uncertainty, if possible Graphical representation of results and sensitivity analysis

Discussion l l

l l l

l

Summary of results Summary of sensitivity of results to assumptions and uncertainties in the analysis Robustness of the analysis Limitations of the study Relevance of the results to specific policy questions or decisions Results of related economic analyses

Specific items included may differ on the basis of the type of economic analysis undertaken. Source: From Ref. 12.

vaccines has shown that CEA is iterative and that as more data become available with routine use of a vaccine or on the consequences of disease, analyses are refined and results may substantially differ.

SUMMARY Economic analyses are an important element in setting priorities for health interventions, and making decisions on vaccine development and vaccine recommendation. Results can provide the economic rationale for the use of scarce health resources on vaccines and vaccination programs, and provide

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guidance on the most effective ways to implement new and current vaccines. The type of economic analysis performed and the components included in the analysis will differ on the basis of the questions being asked. Attention to the issues outlined in this chapter can help to provide the basis for well-designed economic analyses that will be useful to decision makers faced with many alternative health interventions but limited economic resources.

REFERENCES 1. DiMasi JA, Hansen RW, Grabowski HG. The price of innovation: new estimates of drug development costs. J Health Econ 2003; 22:151–185. 2. Wallace G. Presentation to the Advisory Committee on Immunization Practices, October 24, 2007. Available at: http://www. cdc.gov/vaccines/recs/acip/downloads/mtg-slides-oct07/ 15combvac.pdf. Accessed 1/22/08. 3. Davis MM, Zimmerman BA, Wheeler JRC, et al. Childhood vaccine purchase costs in the public sector: past trends, future expectations. Am J Public Health 2002; 92:1982–1987. 4. Institute of Medicine. Financing vaccines in the 21st century. Washington, DC: National Academies Press, 2003. 5. Lieu TA, Cochi SL, Black SB, et al. Cost-effectiveness of a routine varicella vaccination program for U.S. children. JAMA 1994; 271:375–381. 6. Levine OS, Cherian T, Shah R, et al. PneumoADIP: an example of translational research to accelerate pneumococcal vaccination in developing countries. J Health Popul Nutr 2004; 22:268–274. 7. Haddix AC, Teutsch SM, Shaffer PA, et al. Prevention Effectiveness: A Guide to Decision Analysis and Economic Evaluation. New York: Oxford University Press, 1996:1–227. 8. Morrow RH, Bryant JH. Health policy approaches to measuring and valuing human life: conceptual and ethical issues. Am J Public Health 1995; 85:1356–1360. 9. Prosser LA, Ray GT, O’Brien M, et al. Preferences and willingness to pay for health states prevented by pneumococcal conjugate vaccine. Pediatrics 2004; 113:283–290. 10. Johnson FR, Bingham MF. Measuring stated preferences for pharmaceuticals: a brief introduction. Triangle Economic Research, General working paper G-0002, June 13, 2000. Available at: http://www.ter/com/general.html. 11. Naficy A, Rao MR, Paquet C, et al. Treatment and vaccination strategies to control cholera in sub-Saharan refugee settings. JAMA 1998; 279:521–525. 12. Lieu TA, Ray GT, Black SB, et al. Projected cost effectiveness of pneumococcal conjugate vaccination of healthy infants and young children. JAMA 2000; 283:1460–1468. 13. Loomes G, McKenzie L. The use of QALYs in health care decision making. Soc Sci Med 1989; 28:299–308. 14. Murray CJL. Quantifying the burden of disease: the technical basis for disability adjusted life years. Bull World Health Organ 1994; 72:429–445. 15. Nord E. Methods for quality adjustment of life years. Soc Sci Med 1992; 34:559–569. 16. Gold MR, Siegel JE, Russell LB, et al. Cost effectiveness in health and medicine. New York: Oxford University Press, 1996. 17. WHO. World health report 2002: reducing risks, promoting healthy life. Geneva: World Health Organization, 2002. 18. Stone PW, Teutsch S, Chapman RH, et al. Cost utility analyses of clinical preventive services: published ratios, 1976–1997. Am J Prev Med 2000; 19:15–23. 19. Coffield AB, Maciosek MV, McGinnis M, et al. Priorities among recommended clinical preventive services. Am J Prev Med 2001; 21:1–9. 20. Stratton KR, Durch JS, Lawrence RS. Vaccines for the 21st Century: A Tool for Decision Making. Washington, DC: Institute of Medicine, National Academy Press, 1999:1–460.

21. Beutels P, Thiry N, Van Damme P. Convincing or confusing? Economic evaluations of childhood pneumococcal conjugate vaccination—a review (2002–2006). Vaccine 2007; 25:1355–1367. 22. Lieu TA, Capra AM, Makol J, et al. Effectiveness and cost effectiveness of letters, automated telephone messages, or both for underimmunized children in a health maintenance organization. Pediatrics 1998; 101:E3. 23. Sansom SL, Barker L, Corso PS, et al. Rotavirus vaccine and intussusception: how much risk will parents in the United States accept to obtain vaccine benefits? Am J Epidemiol 2001; 154:1077–1085. 24. Lieu TA, Black SB, Ray GT, et al. The hidden costs of infant vaccination. Vaccine 2001; 19:33–41. 25. Weinstein MC, Siegel JE, Gold MR, et al. Recommendations of the panel on cost-effectiveness in health and medicine. JAMA 1996; 276:1253–1258. 26. Russell LB, Gold MR, Siegel JE, et al. The role of cost-effectiveness analysis in health and medicine. Panel on cost-effectiveness in health and medicine. JAMA 1996; 276:1172–1177. 27. Siegel JE, Weinstein MC, Russell LB, et al. Recommendations for reporting effectiveness analysis. Panel on cost-effectiveness in health and medicine. JAMA 1996; 276:1339–1341. 28. Phillips KA, Chen JL. Impact of the U.S. panel on cost-effectiveness in health and medicine. Am J Prev Med 2002; 22:98–105. 29. Mandelblatt JS, Fryback DG, Weinstein MC, et al. Assessing the effectiveness of health interventions for cost-effectiveness analysis. J Gen Intern Med 1997; 12:551–558. 30. Goldie S. Public health policy and cost-effectiveness analysis. J Nat Cancer Inst Monographs 2003; 31:102–110. 31. Brisson M, Edmonds WJ. Impact of model, methodological, and parameter uncertainty in the economic analysis of vaccination programs. Med Decis Making 2006; 26:434–446. 32. Cutts FT, Zaman SMA, Enwere G, et al. Gambian Pneumococcal Vaccine Trial Group. Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomized, double-blind, placebocontrolled trial. Lancet 2005; 365:1139–1146. 33. Sinha A, Levine O, Knoll MD, et al. Cost-effectiveness of pneumococcal conjugate vaccination in the prevention of child mortality: an international economic analysis. Lancet 2007; 369:389–396. 34. Ray GT, Whitney CG, Fireman BH, et al. Cost-effectiveness of pneumococcal conjugate vaccine: evidence from the first 5 years of use in the United States incorporating herd effects. Pediatr Infect Dis J 2006; 25:494–501. 35. Thompson WW, Shay DK, Weintraub E, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 2003; 289:179–186. 36. Thompson WW, Shay DK, Weintraub E, et al. Influenza associated hospitalizations in the United States. JAMA 2004; 292:1333–1340. 37. Widdowson M-A, Meltzer MI, Zhang X, et al. Cost-effectiveness and potential impact of rotavirus vaccination in the United States. Pediatrics 2007; 119:684–697. 38. Tucker AW, Haddix AC, Bresee JS, et al. Cost effectiveness analysis of a rotavirus immunization program in the United States. JAMA 1998; 279:1371–1376. 39. Koopmanschap MA, Rutten FFH, van Ineveld BM, et al. The friction cost method for measuring indirect costs of disease. J Health Econ 1995; 14:171–189. 40. Johannesson M, Karlsson G. The friction cost method: a comment. J Health Econ 1997; 16:249–255. 41. Hornberger J, Robertus K. Cost-effectiveness of a vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. Ann Intern Med 2006; 145:317–325. 42. Bureau of Labor Statistics. Quarterly Census of Employment and Wages. Available at: http://www.bls.gov/qcew/home.htm. Accessed 1/22/2008. 43. Bureau of Labor Statistics.Nonfatal cases involving days away from work: selected characteristics (2003). Available at: http:// data.bls.gov/PDQ/outside.jsp?survey=ch. Accessed 1/22/2008.

Chapter 11: Economic Analyses of Vaccines and Vaccination Programs 44. White T, Lavoie S, Nettleman MD. Potential cost savings attributable to influenza vaccination of school-aged children. Pediatrics 1999; 103:E73. 45. Cohen GM, Nettleman MD. Economic impact of influenza vaccination in preschool children. Pediatrics 2000; 106:973–976. 46. Luce BR, Zangwill KM, Palmer CS, et al. Cost effectiveness analysis of an intranasal influenza vaccine for the prevention of influenza in healthy children. Pediatrics 2001; 109:E24. 47. Nichol KM. Cost benefit analysis of a strategy to vaccinate healthy working adults against influenza. Arch Intern Med 2001; 161:749– 759.

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48. Doubilet P, Begg CB, Weinstein MC, et al. Probabilistic sensitivity analysis using Monte Carlo simulation: a practical approach. Med Decis Making 1985; 5:157–177. 49. Drummond MF. Guidelines for authors and peer reviewers of economic submissions to the BMJ. BMJ 1996; 313:275–283. 50. Lieu TA, Meltzer MI, Messonier ML. Guidance for health economics studies presented to the Advisory Committee on Immunization Practices. November 13, 2007. Available at: http://www. cdc.gov/vaccines/recs/acip/downloads/economics-studiesguidance.pdf. Accessed 1/22/08.

12 An Overview of U.S. Food and Drug Administration Licensure of Vaccines Lydia A. Falk and Norman Baylor Center for Biologics Evaluation and Research, FDA, Rockville, Maryland, U.S.A.

Karen Midthun Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, U.S.A.

INTRODUCTION This chapter provides an overview of the regulatory authority for the licensure of biologics in the United States, as conveyed to the U.S. Food and Drug Administration (FDA). It includes a brief description of the statutory authority and implementation of this authority by the FDA. It describes the role of the FDA in product development, especially vaccines, and how the FDA responds to scientific advancements to ensure a state of the art, scientifically based regulatory approach to these products throughout their life cycle (pre-market, licensure, and post-market).

OVERVIEW OF FDA REGULATORY AUTHORITY The licensure of biologics, including vaccines, is addressed in the U.S. federal laws as specified in the Public Health Service Act (PHS Act) section 351 (1) and the Food, Drug and Cosmetic Act (FD&C Act) (2). These Acts provide the statutory authority by which the FDA, through the Center for Biologics Evaluation and Research (CBER), Office of Vaccines Research and Review (OVRR), conducts its review and approval of vaccine license applications as well as post-licensure regulatory activities. The FDA implements these authorities through regulations, which are codified in the Code of Federal Regulations (CFR). Title 21 of the CFR, parts 600 through 680 (3), contains the regulations specific to the licensure of vaccines and other biological products. In addition to these specific regulations for vaccines, other sections of the CFR are fundamental to the manufacture and development of drugs and biologics. These include regulations on current Good Manufacturing Practice (cGMP) (21 CFR parts 210-211) (4) and the clinical development of investigational drugs (21 CFR part 312) (5). In the development of investigational vaccines in the United States, clinical evaluation must be conducted under the authority of the FDA through the submission of an Investigational New Drug (IND) application. The establishment of an IND allows for the FDA and the vaccine developer to communicate on all aspects of product and clinical development. 21 CFR 312.20 subpart B describes the requirements for an IND, the phases of investigations, the content and format of the IND, as well as the administrative requirements for submitting information to the FDA, including required periodic reporting. To address the need for expediting public access to new drugs and biologics, the US Congress passed the Prescription

Drug User Fee Act of 1992 (PDUFA), which permitted the FDA to collect fees to enhance the review process. PDUFA was reauthorized as part of the Food and Drug Administration Modernization Act of 1997 (PDUFA II), and the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 (PDUFA III), both of which amended the FD&C Act and the PHS Act. The implementation of the PDUFA has resulted in establishing timelines and performance goals for identified review activities conducted by the FDA, provided for additional guidelines for meeting with industry, as well as provided for fees (user fees) to support FDA’s review of marketing applications. The implementation of these timelines and fees is intended to increase the transparency by which the FDA conducts its review and to facilitate the timeliness of product development and licensure. CBER is committed to facilitating public access to safe and effective products. In fulfilling this commitment, CBER plays an active role in the scientific-based review during all stages of clinical development from IND through Biologics License Application (BLA). The stages of clinical development as outlined in 21 CFR 312.21 include phase 1 studies that evaluate primarily the safety of the product in a small number of subjects; phase 2 studies that evaluate the safety and sometimes preliminary effectiveness outcomes in a larger number of subjects; and phase 3 studies that are typically large-scale safety and efficacy studies needed to support licensure (5). Phase 4 studies are conducted post-licensure and are typically designed to gain additional safety data and occasionally to obtain additional efficacy data in broader populations than were studied in phase 3. Throughout all phases of clinical development, safety is a primary focus through the review of the manufacturing and testing, nonclinical toxicity data, as well as all previous human experience. Early in clinical development the data for supporting safety may be limited and therefore it is appropriate to limit the number of subjects in initial clinical testing. As clinical development progresses to phase 2 and especially phase 3, CBER reviewers increasingly focus their review on the robustness of chemistry, manufacturing, and controls (CMC) testing, the study design and objectives with regard to safety and effectiveness, as well as the statistical analysis plans for the proposed clinical protocols.

Chapter 12: An Overview of U.S. FDA Licensure of Vaccines

CBER IMPLEMENTATION OF REGULATORY AUTHORITY In executing its regulatory authority, CBER relies on the regulations as stated in the CFR as noted above. CBER’s regulatory authority covers pre-marketing (investigational status), licensure, and post-marketing activities. A brief overview of CBER’s regulatory activities in each of these areas is described below.

Pre-Market In the United States, the clinical evaluation of a vaccine prior to licensure (i.e., an investigational vaccine) must be conducted under the authority and oversight of the FDA (21 CFR 312) through submission of an IND. The submission of an IND is required to allow the introduction of the investigational product into interstate commerce and its investigational use. In exercising this oversight authority, CBER is required to review the CMC information, nonclinical safety and activity testing, and any relevant clinical testing data regarding the investigational vaccine. The initial submission of the IND triggers the formation of a multidisciplinary review team, which evaluates product manufacturing and testing, nonclinical testing design and outcomes, clinical trial design, statistical design and analysis, as well as assessing the environment and the facility used to manufacture the product. Throughout all stages of product development, safety of human subjects is paramount. Safety assessments are conducted based on CMC considerations, safety signals observed in nonclinical studies and lastly, by careful assessment of safety data being generated from clinical studies. With regard to CMC, CBER requires that sponsors submit adequate information as to the starting materials used in vaccine production, such as information on the microbiologic isolate used to generate the vaccine, all raw materials used, sourcing of animal derived raw materials, as well as adventitious agent testing and thorough characterization of cell substrates used in manufacturing. In addition to focusing on the materials used to produce the vaccine, CBER also requires that information on the manufacturing process, the facility and equipment used to manufacture the vaccine, as well as testing controls be submitted in order to demonstrate control of manufacture and to support the purity and potency of the vaccine. As investigational products reach advanced stages of product development, such as phase 3, additional information should be provided to demonstrate the robustness of the manufacturing process. Consistency of manufacture is a key component to support a licensing application and is critical to demonstrate in phase 3. Demonstration of manufacturing consistency in phase 3 allows CBER to adequately interpret clinical data generated in phase 3 for support of licensure. Early signs of safety concerns may be apparent from nonclinical studies when toxicity is observed in animals. Nonclinical data can provide a safety signal, which can then be specifically monitored during clinical studies. Nonclinical studies may also be helpful in determining the initial dosage and regimen for phase 1 studies as well as support product formulation changes in order to enhance activity or stability of the product. Nonclinical animal studies are often required over the course of clinical development, especially to support the safe use of the product in special populations such as pregnant women or women of childbearing potential. Given the importance of the nonclinical data to support the safe use of the vaccine in humans, careful consideration is needed as to the

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choice of animal model and study design. It is important to discuss such aspects of any nonclinical study with CBER prior to study initiation to ensure that the study is adequately designed to support clinical use. The clinical assessment of a vaccine occurs using a staged approach in which the initial phase 1 study is conducted in limited number of subjects to assess common events related to local and systemic reactogenicity. In phase 1, the study design may include a dose-ranging evaluation of the vaccine-induced immune response to provide a preliminary assessment of the immunogenicity of different dosages of vaccine administered according to the proposed immunization schedule. As clinical development moves to phase 2, larger numbers of subjects are studied to establish a better-defined safety profile for reactogenicity and adverse events. During phase 2, additional immunogenicity or effectiveness endpoints are studied, and these data are used to determine the most appropriate dosage and immunization schedule to take forward into a pivotal phase 3 study. Phase 3 clinical development involves the design, conduct, and analysis of the pivotal study(ies) to support licensure. Given the critical nature of the phase 3 program, CBER encourages applicants to meet with CBER to discuss their proposed phase 3 study as well other aspects of the program, including status of manufacturing validation and consistency. In review of the study protocol, CBER gives careful attention to the sample size, statistical analysis plan, as well as primary and second-study endpoints. All aspects of the study need to be robust, including the laboratory testing to support efficacy endpoint determinations as well as the safety surveillance. Agreement on the phase 3 program status and study protocol is critical to facilitating the path to licensure, given that licensure of the vaccine for the requested indication will be based on the successful conclusion of the phase 3 study. Upon completion of the phase 3 study, applicants often discuss their plans for BLA submission with CBER. This pre-BLA meeting is designed to advance the understanding between CBER and the applicant as to what data or information is to be submitted in the BLA, as well as the requirements for submitting the BLA either electronically or through paper documentation.

Licensure The process by which CBER approves a product for market is through the issuance of a biologics license. The decision to approve a product is based on CBER’s determination that the product is safe and effective, as demonstrated by the data submitted in the BLA, as well as inspection noted below. The regulations covering licensure of vaccines can be found in 21 CFR 600 (3). The BLA submission includes all CMC, nonclinical and clinical data necessary to determine that the product can be made consistently, is stable and meets the criteria for safety, purity and potency. Once the BLA is received, CBER assembles a multidisciplinary review team to review and evaluate all aspects of the application. In addition to reviewing the submission, CBER conducts both a facilities preapproval inspection (PAI) to assess compliance with Good Manufacturing Practices, as well as an inspection of clinical sites (Bioresearch Monitoring; BIMO) where the pivotal study was conducted to assess compliance with Good Clinical Practices (GCP) (6). As part of the CBER deliberations regarding an application, CBER may choose to seek the advice of their

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advisory committees. In the case of vaccines, CBER would request that the Vaccines and Related Biological Products Advisory Committee (VRBPAC) review the product and clinical data and provide comment on the adequacy of the safety and efficacy data submitted in the BLA. Based on the CBER reviews and the advice of the VRBPAC, CBER reaches a decision to approve the application based on demonstrated safety and effectiveness or to inform the applicant of the deficiencies found that preclude approval.

CBER can be through formal submissions to the IND or BLA in annual reports required by regulation. These annual reports provide the status of studies under IND or activities that have occurred relative to a licensed product, including the status of post-marketing commitments. In addition to this required reporting, sponsor/applicants can also seek CBER comment and guidance following submission of data and specific questions throughout the product life cycle. Responses to questions may be provided in writing or may be communicated through meeting with the sponsor/applicant.

Post-Market Once a product is approved, CBER continues product surveillance and oversight throughout the life of the product to ensure that the product continues to meet the standards for purity, potency and safety that were the basis for licensure. Key to this surveillance is the receipt of adverse event reporting through the Vaccine Adverse Event Reporting System (VAERS), a passive reporting system for capturing adverse events postlicensure. In addition, CBER may request that the applicant conduct phase 4 clinical studies to expand the safety database. Such phase 4 studies are agreed to prior to licensure and are considered post-marketing commitments. CBER also obtains information on product performance through lot release information submitted to CBER and the conduct of biennial facilities inspections post-licensure. The review of lot release data and the conduct of CBER-generated confirmatory lot release testing allow for detection of changes in manufacturing performance that may impact the safety or efficacy of the product in distribution. Based on the data received, CBER may engage the manufacturer to better understand the trend and may also conduct an inspection of the facilities as a result of the data review. Changes observed relative to the product can be cross-checked against safety databases such as VAERS to determine whether there are safety or efficacy signals being observed during clinical use of the product. Following licensure, CBER also monitors the distribution of product information to the public. Evaluation of the promotional labeling for a product is evaluated on a routine basis and is assessed for consistency with the product labeling approved by CBER. This surveillance is key to ensuring that accurate, substantiated information is being provided to the public.

Managed Review Process To facilitate a rapid and efficient review environment, CBER has implemented a managed review process (MRP). The MRP allows for CBER staff to have a well-defined process for review throughout the life cycle of the product, starting before and extending through the IND and BLA phases of development and into the post-marketing phase. The MRP addresses all aspects of regulatory activities from public health–based research, management of exports, evaluation of biologics master files, emergency operations, prevention of product shortages, development of regulatory policy, as well as surveillance and enforcement activities (7). The MRP provides a framework by which each review team member can effectively and efficiently review and communicate their observations to CBER staff, as well as interact with sponsors.

Communication with CBER A key component to facilitating product development is open and frequent communication with CBER. Communication with

RESPONDING TO A CHANGING SCIENTIFIC AND REGULATORY LANDSCAPE: CRITICAL PATH INITIATIVES The FDA has recognized the challenges faced by pharmaceutical companies in sustaining a robust product pipeline for innovative medical therapies (8). In the face of new breakthroughs in biomedical science, the need for a greater leveraging of scientific innovation with product development is critical for moving therapies forward. New scientific discoveries need to be matched with new methods for assessing products to enhance the predictability and efficiency of the development pathway. In this regard, the FDA has launched a Critical Path Initiative, which is designed to provide new tools to enhance the product development pipeline. The list of opportunities for initiatives include expediting product development by advancing GMP initiatives, streamlining clinical trials, and developing better evaluation tools such as biomarkers for predicting adverse reactions. The complete Critical Path Opportunities List can be found at the FDA Web site (9). Several areas that may be important in stimulating vaccine product development are noted below.

Biomarkers for Vaccines The demonstration of vaccine efficacy is required under Federal Law. The conduct of large-scale field trials with clinical disease endpoints has typically been the gold-standard for new vaccines. The cost and logistics of conducting such studies was recognized as an impediment to rapid evaluation of vaccine candidates. The Critical Path Opportunities List includes the development of surrogate markers of protection, such as immunogenicity, as an opportunity to improve the speed at which novel products are developed. In addition to efficacy endpoints, the analysis of critical path opportunities also noted that advancements in biomarkers as predictors for vaccine adverse reactions, as well as risks for developing enhanced disease, would also have a major impact on improving the critical path to vaccine development.

Manufacturing Opportunities in the area of improving manufacturing capabilities include the availability of cell lines that are certified free from adventitious agents, which can be used as cell substrates for vaccine manufacture. Use of cell lines for manufacture may improve the consistency of manufacture, reduce adventitious agent testing requirements, and provide a more stable access than use of primary cells. In addition, the development of novel methods for measuring the physical characteristics of products, improved methods for detecting contamination in products, and development of new tools to predict and assess the effect of manufacturing changes on

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product performance are opportunities that could improve the safety, purity and potency evaluations of vaccines.

Table 1 Key Guidance to Facilitate Vaccine Clinical Development Title

Issue date

Improved Disease Models

Draft guidance for industry: characterization and qualification of cell substrates and other biological starting materials used in the production of viral vaccines for the prevention and treatment of infectious diseases Guidance for industry: development of preventive HIV vaccines for use in pediatric populations Draft guidance for industry: clinical data needed to support the licensure of trivalent inactivated influenza vaccines Draft guidance for industry: clinical data needed to support the licensure of pandemic influenza vaccines Guidance for industry: considerations for developmental toxicity studies for preventive and therapeutic vaccines for infectious disease indications Draft guidance for industry: toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials Draft guidance for industry: considerations for plasmid DNA vaccines for infectious disease indications Guidance for industry: FDA review of vaccine labeling requirements for warnings, use instructions, and precautionary information Draft guidance for industry: post marketing safety reporting for human drug and biological products including vaccines Guidance for industry: content and format of chemistry, manufacturing and controls information and establishment description information for a vaccine or related product

9/28/2006

Better predictors of disease models are a major area where additional development is needed. In order to respond to bioterrorism threats, expanded resources are needed for large and small animal models that are suitable for ensuring that products being developed are likely to be safe and effective in humans. Development of models for bioterrorism threat agents such as smallpox, anthrax, as well as naturally occurring threats such as severe acute respiratory syndrome (SARS) and pandemic influenza would greatly facilitate the development of relevant counter measures. Similarly, development of better tissue culture systems for hepatitis C, West Nile, or SARS may improve the product development pipeline by providing more sustainable, screening tools for candidate products directed at those infectious agents. FDA is uniquely positioned to identify these challenges in product development. CBER’s strong research base allows for staff to generate data to support regulatory decisions and is instrumental in identifying and developing solutions for public health regulatory challenges. CBER research impacts a number of aspects of product development including product characterization, potency testing, adventitious agent testing, understanding and optimizing the immune response to vaccines or the disease pathogenesis of organisms, development of standards and methods, as well as developing better clinical data analysis tools. These research activities allow for CBER to be better aligned with the challenges of new technologies or public health questions, as well as to be a major contributor in addressing critical path questions.

CBER OUTREACH TO FACILITATE PRODUCT DEVELOPMENT Although the CFR provides information on the requirements for investigational studies and licensing actions, CBER acknowledges the importance of providing additional guidance and information to vaccine developers on manufacturing, nonclinical testing, and clinical evaluation. Through the issuance of guidance documents, CBER provides its current thinking as to how the requirements of the CFR can be met and provides insight into possible methodologies and approaches to facilitate product development. Although guidance documents are nonbinding, the issuance of these documents allows for CBER to maintain flexibility in addressing specific regulatory challenges, such as cell substrate issues or licensure approaches for biodefense and pandemic influenza countermeasures, and is a key component in CBER outreach to industry. Specific examples of guidance documents that have been issued regarding vaccine manufacture and product development are found in Table 1. A complete list of guidance can be obtained through CBER’s web page (10). Another key component to outreach is CBER’s participation in public workshops and pharmaceutical trade organization meetings to facilitate exchange of information and ideas. CBER staff presentations at these trade meetings have been a highly effective way of communicating new guidances and current thinking in an interactive forum, as well as provide open forums for addressing challenging topics. Examples of workshops in which CBER has been involved include workshops on assessing animal models of efficacy for anthrax and

5/4/2006 3/2/2006

3/2/2006

2/13/2006

4/29/2005

2/17/2005

10/1/2004

3/12/2001

1/5/1999

plague vaccines, toxicity testing approaches for vaccines, and most recently, post-marketing vaccine safety. More information on recent workshops can be found at (11). Given the global nature of vaccine development, the ability of CBER to understand the issues facing our international stakeholders is critical so that international requirements and U.S. requirements could be met in the most efficient manner. CBER maintains international collaborations through representation at international organizations such as the International Conference on Harmonisation (ICH), the World Health Organization (WHO), Pan-American Health Organization (PAHO), and standards development organizations such as Health Level 7 (HL-7). In addition, CBER maintains relationships with other international regulatory authorities such as Health Canada and the European Medicines Authority (EMEA). CBER participation and exchange of information with the international community helps facilitate harmonization and standardization efforts on a more global level.

SUMMARY Product development in the United States is conducted under the authority of the U.S. FDA from early states of investigational use through continued evaluation of product performance post-licensure. Critical to the success of product development is communication between the FDA science-based regulatory reviewers and industry. Outreach is an important aspect of how FDA shares current thinking with vaccine developers and

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is a key component of the FDA’s critical path opportunities for stimulating the vaccine development pipeline.

ACKNOWLEDGMENTS The authors wish to thank Dr Robert Yetter for his assistance in reviewing this manuscript.

REFERENCES 1. Public Health Service Act, July 1, 1944, Chap. 373, Title III, Sec. 351, 58 Stat. 702, currently codified at 42 U.S.C., Sec. 262. Washington D.C.: U.S. Government Printing Office. 2. Food Drug and Cosmetic Act, amended March 2005, currently codified in 21 USC, Sec. 301. Washington D.C.: U.S. Government Printing Office. 3. Code of Federal Regulations, Title 21, Parts 600-680. Washington D.C., Office of the Federal Register, National Archives and Records Administration, 2006.

4. Code of Federal Regulations, Title 21, Parts 210-211. Washington D.C., Office of the Federal Register, National Archives and Records Administration, 2006. 5. Code of Federal Regulations, Title 21, Parts 312. Washington D.C., Office of the Federal Register, National Archives and Records Administration, 2006. 6. U.S. Food and Drug Administration. Available at: http://www. fda.gov/cber/guidance/959fnl.pdf. 7. U.S. Food and Drug Administration. Available at: http://www. fda.gov/cber/gdlns/reviewpdufa.htm. 8. Innovation or Stagnation? Challenge and opportunity on the critical path to new medical products. U.S. Department of Health and Human Services, Food and Drug Administration, March 2004. 9. U.S. Food and Drug Administration. Available at: http://www. fda.gov/oc/initiatives/criticalpath. 10. U.S. Food and Drug Administration. Available at: http://www. fda.gov/cber/vaccine/vacpubs.htm. 11. U.S. Food and Drug Administration. Available at: http://www. fda.gov/cber/scireg.htm.

13 Assuring Vaccine Quality by Strengthening Regulatory Agencies: The Work of the World Health Organization Julie Milstien Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Nora Dellepiane, Lahouari Belgharbi, Liliana Chocarro, and David Wood Department of Immunization, Vaccines and Biologicals, Quality, Standards and Safety Team, World Health Organization, Geneva, Switzerland

INTRODUCTION A functional and robust national regulatory authority (NRA) underlies the sustained supply of vaccines of assured quality. Guarantee of vaccine quality cannot be provided by the manufacturers alone; adequate regulatory oversight is critical to assure this. According to the World Health Organization (WHO), a vaccine is of known good quality (assured quality) (1) provided that l

l

the NRA independently controls the quality of the vaccine in accordance with the six specified functions defined by WHO and there are no unresolved confirmed reports of quality-related problems.

For this reason, WHO has focused its efforts on strengthening regulatory capacity and expertise worldwide. Access to high-quality vaccines in the developing world depends on two additional related activities: providing prequalification services for vaccines and assuring that vaccines targeted primarily at the developing country market are appropriately regulated. WHO’s recent work has been focused on ensuring that all three approaches are followed. This chapter will outline the components of vaccine regulation, define why and how it differs from the regulation of defined chemical medicines, provide a history of vaccine regulation, analyze the components and impacts of the WHO strategy, including the impacts on new vaccine development and access to innovative products, and finally, examine the prospects for the future.

Regulation of Vaccines: Why Are Vaccines Different? It is useful to enumerate the functions involved in vaccine regulation and to consider how they might differ from the regulation of chemical medicines, since these two activities are generally performed by the same branch of a national agency, and sometimes by the same staff. Six basic functions have been defined for an NRA by WHO’s Expert Committee on Biological Standardization (ECBS), a global committee of experts in biologicals regulation convened annually by WHO, which develops norms and standards for vaccine quality and regulation,

as follows (2): (i) marketing approval, (ii) monitoring of adverse effects following immunization, (iii) lot release, (iv) access to a control laboratory, (v) regulatory inspections for monitoring processes and compliance with Good Manufacturing Practice (GMP), (vi) consideration of clinical trial data to demonstrate field performance. These must be supported by underlying legislation and regulations to form a regulatory system. l

Although aspects of these six functions are present for chemical medicines, some of them have different intents. For example, lot-by-lot release of product is inherent in the characteristics of a vaccine as a biological product, synthesized by or related to a living organism, and thus subject to change depending on external conditions such as temperature and pH. Chemical medicines are generally able to be chemically defined and so content and structure can be determined simply by physical and chemical tests; therefore, lot release is less important. In the case of clinical trials, outcome measures will differ considerably. For vaccines, these are generally directed at immunological response and/or efficacy in preventing disease, while for chemical medicines, key outcomes could be concentrations at the site of activity and clearance times, as well as improvement in clinical status. For all products, however, a system of marketing authorization, compliance with GMP, post-marketing surveillance of performance, and some kind of testing protocols will be needed.

Brief Description of WHO Inputs As noted earlier, WHO has used three approaches to assure the quality of vaccines in all countries. These include (i) an initiative to strengthen NRAs; (ii) the prequalification process, important for countries for which vaccines are not procured directly or produced within their borders, but increasingly having more far-reaching impacts; (iii) an initiative to ensure the appropriate regulation of innovative vaccines destined for the developing market. These three activities are firmly rooted in the need for strong regulation, and all depend on the normative activities of WHO, that is, the development of written and physical standards.

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First, written standards, developed by groups of experts and adopted through the ECBS, establish regulatory requirements for production and quality control criteria to assure safe and effective vaccines in immunization programs. Specific examples include new written standards for nonclinical and clinical evaluation of vaccines (3); and vaccine-specific guidelines that can serve as a basis for national regulatory decisions, such as a new written standard for human papillomavirus (HPV) vaccines adopted in 2006 by the ECBS (4). Recently developed WHO documents for specific vaccines also include guidance for clinical evaluation of such products, in contrast to earlier documents, that dealt only with production and quality control. These WHO recommendations, if adopted by member countries, can become part of their national requirements for specific vaccines. Second, physical standards developed by WHO are used globally to standardize and validate the methods used to test vaccines. These can be International Biological Standards or International Biological Reference Reagents (5). The written and physical standards provide the basis for the regulation of vaccines as biological products.

HISTORY OF EFFORTS TO ENSURE VACCINE QUALITY Development of Vaccine Regulation in the United States (6) The U.S. Food and Drug Administration (FDA), the agency responsible for regulatory oversight of vaccines in that country, recently celebrated its 100th anniversary, measured from Congress’s passage of the Biologics Control Act on July 1, 1902. This Act was a response to the deaths of 13 children in St. Louis in 1901 after receiving diphtheria antitoxin that had been accidentally contaminated with tetanus. A similar contamination of smallpox vaccine resulted in the deaths of nine children the same year. The Biologics Control Act charged the Laboratory of Hygiene of the Marine Health Service with the regulation of biologicals. Under the Act, the Laboratory issued regulations to ensure safety, purity, and potency; established standards; and issued licenses for smallpox and rabies vaccines, and later for other biological products. It was renamed the National Institute of Health in 1930. All issued regulations and standards were codified in the Public Health Service (PHS) Act of 1944 (7). A milestone in the regulation of vaccines followed the ‘‘Cutter incident’’ in 1955, when 260 cases of polio and 11 deaths resulted from the use of incompletely inactivated polio vaccine manufactured by Cutter Laboratories. The Surgeon General then suspended all polio vaccinations pending a review of vaccine testing procedures and inspections of all manufacturing facilities, which resulted in stricter standards and tighter control. On July 1, 1972, the Division of Biologics Standards was moved from the National Institutes of Health to the FDA, because of a failure to initiate an effectiveness review, so that the provisions of the Food and Drug Control Act as well as those specifically designed for biologicals (section 351 of the PHS Act) applied to regulatory oversight of vaccines. Biological products are currently overseen by FDA’s Center for Biologics Evaluation and Research.

Standardization Activities of the ECBS Section 351 of the PHS Act defines a biological product as a ‘‘virus, therapeutic serum, toxin, antitoxin, vaccine, blood,

blood component or derivative, allergenic product, or analogous product, . . . applicable to the prevention, treatment, or cure of diseases or injuries of man’’ (8). These products have been treated differently than chemical medicines because they are difficult to characterize, often contaminated, and assuring potency, consistency, and safety poses particular challenges. They involve starting materials of biological origin and often need biological testing systems (9). The issues of inherent variability and of measuring potency led to the use of an international standard or reference reagent in quality control tests. The League of Nations recognized the need for biological standardization; this led to the creation of the Permanent Commission on Biological Standardization (10). This work was eventually taken over by the ECBS. Established in 1947, the ECBS has overall responsibility for setting written standards and establishing reference preparation materials. Members of the ECBS are scientists from NRAs, academia, research institutes, and public health bodies. These scientists act as individual experts and not as representatives of their respective organizations or employers. The decisions and recommendations of the ECBS are based entirely on scientific principles and public health considerations. In the early days of vaccine development, as has been seen, vaccines as impure biological products underwent tests, usually in animals, as the sole means of ensuring that the product complied with specifications. Despite biological standardization, they are still not ideal. Two developments have changed the role of testing in vaccine regulation (11): (i) the evolution of concepts of regulation, with more emphasis on assuring consistency of production, through GMP compliance, and more attention to how clinical data are obtained and assessed, both prior to and after marketing (12) and (ii) the changes in the products themselves, which affect the type of testing that is done.

Assuring United Nations Agency Vaccine Supply After the Expanded Program on Immunization (EPI) was started in 1974, vaccines were provided either by multinational manufacturers or national vaccine producers exporting products through United Nations (UN) procurement agencies, including UNICEF and the Pan-American Health Organization (PAHO) Revolving Fund (RF) (13,14).a Vaccine quality was assumed to be acceptable, but even so it was noted that ‘‘PAHO/WHO screen manufacturers offering vaccines for EPI use and, where possible, review protocols of the specific lots submitted for sale’’ (15). In addition, much emphasis was placed on the national testing of vaccines or the use of WHO or PAHO testing centers (16). At its 84th meeting in June 1980, the PAHO Executive Committee urged all Member States to strengthen their respective laboratories for vaccine testing (17). In 1981, WHO’s ECBS published its first guideline on national control of vaccines (18), mandating a ‘‘national control authority’’ for all countries, the responsibilities of which would a

The PAHO RF started buying vaccines in 1978 for 1979. Suppliers of OPV, DTP, TT, measles, and BCG vaccines included Torlak (Yugoslavia), Merieux (France), SmithKline-RIT (Belgium), Connaught (Canada), Japan BCG (Japan), Evans Medical/Glaxo (U.K.). A search of the UNICEF archives failed to provide specific information such as that for the PAHO RF cited here; however, it is safe to assume that WHO activities in the area of quality assurance were comparable for both and also that the suppliers were similar for both procurement agencies.

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differ according to capacity and need. Such an authority was recommended to be empowered to establish or recognize requirements for acceptability of products, establish standard preparations for biological testing, license manufacturers of biological products, and establish facilities needed to implement the requirements. Again, most of the emphasis was placed on testing. In 1987, WHO published its first prequalification requirements (19), supplemented the following year by a modification for bacille Calmette-Gue´rin (BCG) vaccine (20), because of a need for annual clinical trials to establish the consistency of production of this vaccine. These early documents focused on file review, review of consistency of production (generally by review of summary lot protocols and by testing, but supplemented with clinical trials for BCG), and inspection. The procedure was revised in 1988 and reissued in 1989 (21) to take into account the need for the national control authority of the country where the vaccine was produced to exercise its functions.

The Role of NRAs in Prequalification The latest version of the prequalification process, which was updated in 1996 and 2002, and most recently in October 2004 (22), specifically emphasizes the need for the NRA to be fully functional as this is a critical aspect of the prequalified status of a product. Thus, although the primary reason for developing the prequalification process was to advise UN procurement agencies on purchase, its second major impact has been to leverage compliance to standards of NRA function. WHO’s activities in strengthening regulatory authorities to meet the definition of functional will be detailed below. Even if the regulatory process has been thorough and effective, WHO needs to be sure that the product consistently meets the specifications of the UN tender document, that it has been tested in the appropriate target population(s) at the appropriate schedule(s) and with the appropriate concomitant products. Products that may be approved for use in an industrialized country may be recommended for a different age group, a different indication, or using a different immunization schedule than those in most developing country immunization programs. In addition, as mentioned above, there are continuing responsibilities of both the manufacturer and the NRA regarding requirements for reporting of changes in the process, the product, and the facility, and especially for reports of adverse reactions, as well as ongoing lot release responsibility. WHO needs to be sure that these will be honored. The process does not differ depending on the nature or reputation of the NRA, although those NRAs that are less developed may take more time to successfully pass the assessment process. Through this process, NRAs have been increasing in competence.

NRA STRENGTHENING Background WHO has a mandate to assist countries in using vaccine of assured quality (2,23–26) by promoting effective vaccine regulation systems and advising UN agencies on the acceptability in principle of vaccines for immunization programs (prequalification). WHO has been actively engaged in NRA strengthening since 1997, insisting on competent regulatory oversight and the need for a single standard of quality in all countries and

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recommending that only vaccines of assured quality be considered for use in national immunization programs. There are three primary ways a country gains access to vaccines: purchasing through centralized procurement such as UNICEF or WHO, procuring vaccines directly or through other types of purchasing agents, or producing vaccine locally. On the basis of the WHO/UNICEF joint reporting monitoring form launched in 1999, as of December 31, 2006, of 193 WHO member states, 48 (25%) produce vaccines, 63 (32%) procure their vaccines, and 82 (43%) use UN agencies such as UNICEF to source their vaccines.

WHO Process to Strengthen NRAs for Vaccines To ensure all countries have access to vaccines of assured quality, and that the quality is maintained up to the time the vaccine is administered in the target population, a five-step capacity-building program has been developed. These five steps are developing the benchmarks for national systems; assessing these systems against published indicators; developing an institutional development plan (IDP) to address any gaps found; implementing the plan, using, among other things, the Global Training Network (GTN); and monitoring the impact of the interventions. Primary efforts have focused on strengthening the capacity of NRAs in countries producing vaccines, and then on those that procure their vaccines, so that these will also be of assured quality. Countries do not need to establish a new regulatory authority just for vaccines. As the majority of the functions are similar to those exercised by drug regulatory authorities, the necessary additional expertise can be gained through appropriate use of advisory committees, by technical support, and through training (26). Thus the approach is to build on existing drug regulatory authorities. Figure 1 explains the different functions within a national regulatory system and shows the interdependence of the various regulatory functions during the pre- and post-marketing phases. Different institutions (National Control Laboratory, GMP inspectorate, NRA, immunization program, ethics committees, etc.) and different types of expertise (regulators, epidemiologists, GMP inspectors, scientists, etc.) may provide inputs. National systems need strong coordination and a documented process to use all these inputs effectively for regulatory oversight. Step 1: Developing the Benchmarks In 1990, WHO estimated that only 15% of the 69 vaccineproducing countries at the time had an independent and functional vaccine regulatory system. Since 1995, starting with WHO’s GTN, WHO began to focus on NRAs, national immunization programs, pharmacovigilance units, and drug inspectorates involved in regulation of vaccines. In 1997, work started to develop an assessment tool to benchmark functional national regulatory systems. This was developed with the input of 38 countries and discussed in intercountry workshops. The tools were reviewed regularly to refine them and harmonize the assessment process. The NRA assessment tool that was developed in 1997 was subsequently revised in 1999, 2001, 2002, and 2004 using informal consultations of international regulatory and vaccine experts from functional NRAs recruited from a wide geographic distribution (Americas, Europe, Asia, and Africa) (Table 1). Revisions conducted in 2001 and 2002 were also used for developing a joint NRA assessment tool for drugs, vaccines, and medical devices.

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Table 1 Countries Participating in the Review of Assessment Tools Year

1999

2001

2002

2004

Country experts

Argentina Canada Germany Iran Japan Indonesia France Senegal Switzerland Tunisia United Kingdom United States

United States Russia Indonesia China Cuba Belgium Australia France Thailand India

United States Russia Indonesia France Cuba Belgium Australia Thailand India China United Kingdom

United States Belgium South Africa Indonesia India France Russia Australia Cuba China United Kingdom

Figure 1 The vaccine regulatory process.

Step 2: Assessing NRAs Against Indicators Since 2001, WHO joint assessments of regulatory systems for drugs, vaccines, and medical devices have been conducted in 20 countries. For assessment of vaccine NRA functions, Table 2 shows the total number of countries assessed for vaccine NRA function: 86 of 193, or 45%.

Table 2 NRA Assessments Conducted Against Indicators

WHO region

Number of national regulatory authorities assessed

Total countries

Percentage (%)

Step 3: Addressing the Gaps As a result of these NRA assessments, existing gaps in the regulatory system can be identified and translated into an IDP, which, after endorsement by the corresponding government, can help address the gaps through training and other capacitybuilding activities.

African American Eastern Mediterranean European Southeast Asian Western Pacific Total

28 5 13

47 35 22

60 14 59

22 10 8 86

52 11 26 193

42 91 31 45

Step 4: Implementing the IDP The GTN is a WHO initiative, started in 1995, which provides training to NRA staff in the six regulatory functions through

13 training centers distributed throughout the world. The training curricula are regularly updated to meet the needs of the audience as they increase their level of understanding and

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expertise. Curricula cover all the regulatory functions, and new training curricula are developed as needs arise. Currently a training curriculum on testing of conjugate vaccines is being developed and the training course on lot release is being updated. A course on clinical evaluation of vaccines has been reformatted to focus on authorization of clinical trials and a new course on Good Clinical Practice (GCP) inspection has been developed by the Developing Countries Vaccine Regulators Network (DCVRN) (see below). The impact of training in raising the expertise of the NRAs and the progress of NRAs in developing their systems is measured through follow-up visits or reassessment at regular intervals. More than 2000 staff have been trained through intercountry courses that were conducted in all WHO regions and involving experts from 80 countries. Step 5: Monitoring the Impact GTN follow-up workshops and visits were conducted to monitor implementation of IDPs. The GTN was helpful in building a roster of regulatory experts for conducting assessments and follow-up visits and providing decentralized training. There is now a core of technical expertise from 90 countries available to other countries upon request. There is an Advisory Committee on Training that meets every two or three years to review progress in training as well as to identify improvements for strengthening NRAs. Several regulatory authorities in industrialized countries that have already implemented a strong and efficient regulatory system have reliable skills and expertise to assist developing countries. Countries from the European Union (France, Belgium, Germany, United Kingdom, Italy, the Netherlands) as well as Russia, United States, Canada, and Australia have contributed actively to this process. On the basis of assessments, WHO can determine the impact of the NRA strengthening initiative by monitoring the number of doses of assured quality. Currently, of the approximately 12.3 billion doses of vaccine in use in the world, 8.6 billion (70%) is of assured quality (Fig. 2).

Figure 2 Proportion of vaccines of assured quality by region, 2007.

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The NRA strengthening initiative has been largely responsible for the emergence of vaccine manufacturers from developing countries on the global market; in addition, the converse is true: The existence of emerging manufacturers of vaccines has triggered the strengthening of the NRAs of their countries so that production is properly regulated.

PREQUALIFICATION OF VACCINES The vaccine prequalification system was formally put in place within WHO in 1987 to provide advice to UN procuring agencies on the quality, safety, and efficacy of vaccines for purchase. Currently, a total of 124 countries are served by UN procurement agencies, 90 through UNICEF Supply Division, and 34 through the PAHO RF. This accounts for approximately 58% of the total population receiving vaccines of assured quality. Because of the importance of oversight of the manufacturer’s NRA for prequalification of a product, a new policy endorsed by an expert committee in 2004 requires a mandatory assessment of NRAs in all the countries for which prequalified vaccines are listed and in countries where manufacturers intend to apply to WHO for vaccine prequalification. The steps of the current prequalification process, described in a WHO publication (22), begin with an assessment of the overseeing NRA, to ensure that it is functional. If it is, WHO reviews the manufacturer’s product summary file, which describes the product, the production process, the facilities, the quality system, and clinical and nonclinical data generated to demonstrate its safety and utility for the intended purpose in the target group. The written standards developed by the ECBS are one of the bases for developing specifications for the procurement tender document. WHO provides independent testing to confirm consistency of final product characteristics and to ensure that the product meets the specifications in the relevant tender, and organizes a site visit to the manufacturing facilities to assess compliance with GMP and to verify the information in the file. Agreements are reached with the manufacturer and the NRA on continuing responsibilities, reporting requirements, and shipping and packaging specifications. Figure 3 shows the current dependence of the prequalification process on NRA status. Once prequalification status has been conferred, the continuing oversight of the prequalified vaccine falls under the responsibility of the relevant NRA. When the vaccine is reassessed, a process that happens at prescribed intervals to maintain prequalification status, by a WHO team of experts, generally NRA representatives participate either as observers or as team members during the site visit. The information gathered during the reassessment both from review of an updated product summary file and at the time of the site visit serves as feedback on the NRA performance with regard to the oversight of the specific product and leverages further improvement. In the past, most prequalified vaccines were produced in industrialized countries. An outcome of the prequalification process is that it sets up an independent and unbiased method to evaluate vaccines proposed for purchase no matter where they are produced—thus allowing emerging suppliers to compete on the international market. The result of this is that the number and percentage of vaccines, both traditional and new, coming from emerging suppliers, whose prices may be lower, and who may be able to supply basic vaccines in larger

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Figure 3 Schematic diagram of the role of the NRA in the prequalification process for vaccines. Boxes that are shaded indicate activities where the functioning of the NRA is key. Abbreviation: NRA, national regulatory authority.

Table 3 Statistics on Sources of Prequalified Vaccines

Year 1986 1996 2006

Number of vaccine types 6 13 24

Number of suppliers 13 18 22

Percentage (%) from emerging economies 13 37 55

quantities, have increased, contributing to the security of vaccine supply worldwide (27). Table 3 shows this development. Table 3 also shows the increase in number of prequalified vaccine types, and thus of prequalified products in the last 10 years. Table 4 shows the evolution of types of vaccines submitted for evaluation between 1986 and now as well as the projected submissions for 2007 and after. Table 4 shows most activity dedicated to traditional vaccines in the past, as expected, but still ongoing, as emerging suppliers take over this market. Vaccine security is a high priority for UN purchasing agencies and is therefore high priority for WHO prequalification. In recent years, developing country manufacturers have also developed diphtheria toxoid/ tetanus toxoid/whole-cell pertussis–based combinations, which

are replacing the use of diphtheria-tetanus-pertussis (DTP), Hepatitis B, and Haemophilus influenzae type b administered separately. The evaluation of combination vaccines is a high priority for WHO and this evolution is also reflected in Table 4. In addition to sustaining the availability of vaccines already introduced in routine immunization programs and those required for outbreak response (meningococcal, cholera, etc.), accelerating introduction of new vaccines that have recently become available is of high priority, particularly for the PAHO RF, particularly rotavirus and pneumococcal conjugate vaccines. As NRAs become stronger, it is expected that the prequalification process will allow more reliance on the relevant NRAs, tailored according to factors such as the experience of the manufacturer and NRA, and to the level of risk that has been seen with similar products—for example, linked to incidence of reported adverse events following immunization or to the experience of the vaccine producer in supplying prequalified products. Thus the process will be more focused on novel vaccines. Products will be classified in one of the following three categories: l

Category A. High risk: products submitted by manufacturers with limited or no experience with the prequalification process or with the product in question, and/or the NRA is borderline with respect to functionality.

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Table 4 Percentage of Vaccines of Each Type Expected for Prequalification Evaluation Type of vaccine b

Combination vaccines (%) Novel and underused (%)c Traditional (%)d

1986

1996

2006

2007a

– – 100

– – 100

8 7 83

30 20 50

2008a 43 4 14

2010 and aftera – 80 20

a

Projected. Diphtheria toxoid/tetanus toxoid/whole-cell pertussis–based combination vaccines. c Rotavirus, human papillomavirus, pneumococcal conjugate, meningitis conjugate, HIV, malaria, Japanese encephalitis, Haemophilus influenzae type b, cholera, rabies vaccines, etc. d Measles-mumps-rubella (MMR) group, diphtheria-tetanus-pertussis (DTP) group, oral poliovaccine (OPV), inactivated poliovaccine (IPV), yellow fever, meningitis polysaccharide, pneumococcal polysaccharide, Hepatitis B. b

l

l

Category B. Medium risk: manufacturer has at least one other prequalified vaccine and has experience with the product in question (although it is not prequalified), or manufacturer is new to the system but is supported by a joint venture with a well-established manufacturer, and NRA is functional. Category C. Low risk: Manufacturer is well established, more than one product prequalified, and may or may not have experience with product in question but has good research and development infrastructure. NRA is functional.

Products in categories A and B will be thoroughly assessed and monitored (as per current procedure). Products in category C will be subject to a lighter process with strong reliance on the NRA responsible for the product regulatory oversight. The prequalification of new vaccines poses additional challenges from the regulatory point of view, not only in terms of evaluation of the product as such, but also because some of these vaccines are likely not to be used in the country of manufacture and therefore the responsible NRA would not have first-hand post-marketing surveillance data to monitor their safety and efficacy. This is a serious problem that affects the prequalification system. To address this, WHO is establishing a global network of sentinel countries having strong postmarketing surveillance systems to monitor the safety profile of recently introduced vaccines over a specified period to increase the data in product safety profiles. One of the constraints for new vaccines produced in the industrialized world is to ensure that clinical safety and efficacy data are available for all areas for which its use is intended, at the prescribed schedule and with the prescribed concomitant vaccines. In such a case, WHO must evaluate the clinical efficacy data carefully, and in some cases, may request more extensive clinical data. An example of this is the rotavirus vaccine, Rotarix1 (GlaxoSmithKline Biologicals, Rixensart, Belgium), manufactured by GSK and recently (February 2007) added to the list of prequalified vaccines. In this case, because of the limited availability of clinical data in all areas of the world, WHO added a statement on their website (28) indicating that safety and efficacy data were available only for Latin America and Western Europe. The list of prequalified products, which is posted on WHO’s website (28) along with the names of the suppliers, and of their NRAs that have been thus assessed and found fully functional (29), provides a support to countries that are procuring vaccines directly, but which may not yet have the capacity to make these decisions on their own. A guideline, Expedited Approval of Vaccines Used in National Immunization Programs,

which was considered by the ECBS in 2006, provides guidance to such countries on using the existence of prequalified status for fast-track approval of vaccines in their countries. This should relieve the burden on both NRAs and manufacturers, but still ensure that countries can procure vaccines of assured quality.

NEW REGULATORY PATHWAYS The pace of new vaccine introductions will increase dramatically. As new vaccines have been developed, the need for a new response to their regulation has become clear, for three reasons.

1.

2.

3.

Because many products will be first manufactured and approved for marketing in industrialized countries, new approaches are needed to ensure appropriate regulatory oversight for them. It was initially assumed that the regulatory oversight of a strong NRA would be sufficient, and that target countries could just rely on their regulatory decision. However, this is not always true. For example, in many cases the product would not be used in the country of manufacture, or, if it were, it might be used for different indications, or at a different schedule, or in a different epidemiological setting. Other issues are appropriate clinical trial oversight, no matter in which country the trials will be held, as well as appropriate risk-benefit considerations for the regulatory process. All these differences raise issues that could interfere with the appropriate regulatory decision being made by one country for a product to be used in another. Because of different levels of benefit-risk ratio, products that might be very useful in a developing country setting might never be approved in their country of manufacture (30). As more emerging suppliers are involved in the development of innovative vaccines, the requirement for a fully functional NRA will put pressure on their national regulatory systems. In many cases, countries being targeted for novel vaccine introduction do not have the full competence and expertise required to assess the quality, safety, and efficacy of the vaccines they will introduce. As manufacturers enter the market with products that can be used in developing countries, there has been a greater need for clinical trials conducted in the endemic countries themselves. The information thus collected contributes to the prequalification process, and to the availability of vaccines of importance to developing countries. However, until recently, the host country was not intimately involved in these clinical trials except for reviews by a local ethics committee.

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This section will examine some of the responses that have been developed to address these issues.

Regulation of Products Manufactured for Use Outside the Country of Manufacture A promising approach is the use of a Scientific Opinion by the European Medicines Authority (EMEA) rather than a Marketing Authorization, referred to as Article 58 (31), which is for medicinal products, including vaccines, that will not be marketed in Europe; this is contingent on WHO, confirming the suitability of the vaccine for such process. The Scientific Opinion is delivered following a procedure that mimics exactly the one followed to grant a marketing authorization for products destined for the European market, but also provides for local inputs, considering the relevant epidemiology and context. This innovative process has been designed for use in prequalification, but it has not yet been tested in an actual decision situation. Vaccines produced in other industrialized countries where they are not used may also be granted a license exclusively for export purposes. This ‘‘export license’’ provision is already in use, for example, by the FDA, but its application to a global vaccine procurement situation and to the prequalification of a product seems difficult. Finally, manufacturers may choose to apply for initial license of novel products in those countries where they intend to market the vaccines rather than, as before, initially seek licensure from the country of manufacture. This was the initial strategy used for the new rotavirus vaccine, Rotarix.

Developing Country Networks to Strengthen Regulatory Decision Making To strengthen developing country capacity to innovative vaccine regulation, mostly on the clinical evaluation aspects, WHO has established networks of regulators and ethics committees in developing countries, for example, the Developing Country Vaccine Regulator’s Network (DCVRN) and the African Vaccine Regulator Network (AVAREF). More advanced NRAs, especially those from the country of manufacture of novel vaccines, are collaborating with these networks, by sharing their expertise during scientific sessions and providing guidance for decisions about clinical trial applications in developing countries (32). It is acknowledged that the legal framework for formal collaboration among NRAs poses challenges, but many agreements have been established to facilitate this process and there is a trend to find innovative options to promote inter-agency consultations. The DCVRN has nine member countries, Cuba, Brazil, China, India, Indonesia, Russia, Korea, South Africa, and Thailand, from five different regions. Its mission is to promote and support the strengthening of the regulatory capacity of NRAs of participating and other developing countries for evaluation of clinical trial applications (including preclinical data and product development processes) and clinical trial data for registration purposes, through expertise and exchange of relevant information. WHO as the Secretariat has facilitated expert participation in scientific sessions organized within the framework of DCVRN for topics relevant to the clinical evaluation of new vaccines including a new tuberculosis vaccine, a vaccine against HIV/AIDS, and HPV, rotavirus, typhoid, Japanese encephalitis, and dengue vaccines. A report, DCVRN’s considerations on clinical evaluation of vaccines, reflects the main issues identified for specific vaccines discussed in the scientific

sessions, including recommendations to review existing guidelines or develop new guidance. Thus members can benefit from the opportunity to share experience, challenges, and views and use the consultation with other NRAs as an ad hoc advisory group. The network members have identified areas that need coordination and support from WHO, and have provided support to regional initiatives through sharing of regulatory procedures, funding of expert staff to support other countries, or taking a lead role in the facilitation of capacity-building activities relevant to clinical evaluation of vaccines. DCVRN members have also initiated the development of new regulatory processes, for example an IND-like system that is being tested on a pilot basis by some of them, as well as co-inspections of clinical trial sites, aiming at future mutual recognition of GCP inspection reports. There are challenges specific to certain regions. In Africa, the ‘‘regional approach’’ to address vaccine regulation issues was initiated by WHO in 2005, establishing AVAREF in 2006. It gathers representatives from NRAs and Ethics Committees from 19 countries identified as targets for clinical trials and introduction of novel vaccines. The objectives are to facilitate access to information on clinical development plans for priority vaccines in the region, to provide a source of expertise to support them in their regulatory review processes, to identify the need for regulatory documents, training, and guidance and to foster collaboration between regulatory agencies of countries that produce vaccines and those of countries where the vaccines are clinically tested. There is also potential to develop model and those where vaccines are harmonized procedures and joint reviews and inspections of clinical trials. The DCVRN and AVAREF have proven to be efficient as opportunities for interaction with advanced NRAs like EMEA and FDA, who support these initiatives by actively sharing their expertise and by their openness toward productive discussions that may lead to future agreements.

Strengthening Clinical Trial Capacity Although most phase I clinical trials are done in the country of manufacture of the vaccine, it is possible that for some vaccines these are conducted in developing countries. Therefore, regulatory capacity in the areas of preclinical, nonclinical product review and in protocol review for initial phase I testing of new vaccines needs strengthening. If developing countries are targets for phase II or phase III trials, the challenges are also significant. Often clinical studies are conducted in developing countries with no experience in regulatory oversight of vaccine clinical trials. Nevertheless, any capacity-building process must not cause undue delays, which could negatively impact the clinical development of a public health priority vaccine. DCVRN members have developed a new training course on GCP inspections, which has been incorporated in the Global Training Network and delivered by the faculty from two member countries (South Africa and Indonesia). They have also developed a checklist for regulatory inspections of clinical trials, which has been translated into French and distributed to countries in different regions. The material was further developed and finalized for delivery by the GTN. In Africa, support has extended to the regulation of clinical trials. For example, the Meningitis Vaccine Project of Program for Appropriate Technology in Health (PATH) has given consent to WHO to use the plan for clinical trials as an opportunity to build regulatory capacity in target countries.

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Knowing in advance what countries would be involved in clinical trials has allowed planning activities such as training and development of regulatory procedures, preparing for the submission of clinical trial applications, and subsequently, with support of experts, the joint review of the dossiers and joint inspection of the clinical trials in selected sites. A joint review of a clinical trial application of a Malaria vaccine, involving seven African countries, has also been facilitated by WHO and there are more opportunities for similar activities in the new future. The Rotavirus Vaccine Project of PATH has also supported activities coordinated by WHO, leading to the strengthening of regulatory capacity for the evaluation of registration dossiers, in countries in Asia and Africa. AVAREF members have embarked in a new initiative that involves the integration of ethical review, regulatory oversight and registration of clinical trials. Although AVAREF was established to deal with vaccine issues, this new project involves clinical trials of medicines in general and aims at strengthening regulation of clinical trials and enhance transparency regarding the existing trials Africa, thus promoting the high quality of data resulting from them. AVAREF members are working on the development of regulatory guidance documents relevant to clinical trials that will be proposed for harmonized adoption. Joint review and joint inspections involving NRAs and ethics committees of countries participating in multicenter clinical trials have been facilitated by WHO and have contributed to the strengthening of the regulation of clinical trials.

LONG-TERM PROSPECTS FOR NRA STRENGTHENING ACTIVITIES Although WHO’s intent is that all countries should sustainably develop appropriate NRAs by 2010, there will continue to be a need for NRA strengthening activities. There are three reasons for this.

1.

2.

3.

The need for all countries to assure the quality of the vaccines they are using. The activities of WHO’s NRA Strengthening Initiative, supported by the World Health Assembly Resolution (33) have had enormous impact, but there are still countries where vaccines are produced, which do not have the ability to assure the quality of those vaccines. WHO aims to address this issue and strengthen these NRAs, with the intent that if manufacturers cannot meet quality standards they will be closed down by the NRAs. Those countries would then need an alternative source of vaccines. Countries that are procuring vaccines directly also need to have NRAs functioning to a certain level; even countries receiving prequalified vaccines through UNICEF or the PAHO RF need some regulatory functions. The continuing development of viable regulatory strategies for vaccines intended for prequalification. Although many emerging suppliers are located in countries with developing regulatory authorities, most of these to date are unequipped to deal with innovation. Innovation in the regulatory context covers a wide range of activities covered above. As the characteristics of vaccines change, as biotechnology is brought more strongly to bear in vaccine production, new approaches to regulation will be needed, and these approaches will need to be transferred to all countries. The continuing need for international procurement processes and thus the prequalification process. For the foreseeable future, the need will continue for international procurement of vaccines, both because of the lack of

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capacity for procurement in some countries and because of the advantages of an international procurement process in terms of price and selection of vaccines, coupled with the advantages to manufacturers of limiting transaction costs for products with low profit margins; thus; the need for prequalification will continue. Accordingly, WHO activities to build NRA capacity will remain important to support the prequalification process. Note added in proof: data collection for this paper ended in 2007.

REFERENCES 1. World Health Organization. Vaccine Supply and Quality Unit. GPV Policy Statement. Statement on vaccine quality. 1997. WHO/ VSQ/GEN/96.02.Rev.1. 2. WHO Expert Committee on Biological Standardization. Fortysecond Report. Guidelines for national authorities on quality assurance for biological products. Technical Report Series 1992, No. 822, Annex 2. 3. WHO Expert Committee on Biological Standardization. Fiftyfourth Report. WHO guidelines on nonclinical evaluation of vaccines. Technical Report Series 2005, No. 927, Annex 1. Available at: http://www.int/biologicals/publications/trs/areas/vaccines/nonclinical_evaluation/en/index.html. Accessed March 30, 2007; WHO Expert Committee on Biological Standardization. Fifty-second report. Guidelines on clinical evaluation of vaccines: regulatory expectations. Technical Report Series 2004, No. 924, Annex 1. Available at: http://www.who.int/biologicals/publications/trs/areas/vaccines/clinical_evaluation/en/index.html. Accessed March 30, 2007. 4. WHO Expert Committee on Biological Standardization. Guidelines to assure the quality, safety and efficacy of recombinant human papillomavirus virus-like particle vaccines. 2006, WHO/ BS/06.2050. Available at: http://www.who.int/biologicals/ publications/trs/en/index.html. Accessed March 30, 2007. 5. WHO Expert Committee on Biological Standardization. Fifty-fifth report. Guidelines for the preparation, characterization, and establishment of international and other standards and reference reagents for biological substances (Revised 2004). Technical Report Series 2006, 932, Annex 2. 6. CBER Vision Newsletter, Special Commemorative Issue, July 2002. Available at: http://www.fda.gov/cber/inside/centnews. htm. Accessed March 30, 2007. 7. Baylor NW, McVittie LD. Changes in the regulations for vaccine research and development. The Jordan Report 20th Anniversary. Accelerated Development of Vaccines. US Department of Health and Human Services, National Institute of Allergy and Infectious Diseases, 2002:45–50. 8. FDA. Draft document concerning the regulation of placental/ umbilical cord blood stem cell products intended for transplantation or further manufacture into injectable products. Docket No. 96N-0002. December 1995. Available at: http://www.fda.gov/ cber/bldmem/cordstem.txt. Accessed March 30, 2007. 9. Biological Standardization and Control. A Scientific Review Commissioned by the UK National Biologicals Standards Board. Geneva: World Health Organization, 1997. WHO/BLG/97.1. 10. Hilleman MR. Vaccines and the Vaccine Enterprise: historic and contemporary view of a scientific initiative of complex dimensions. The Jordan Report 20th Anniversary. Accelerated Development of Vaccines. US Department of Health and Human Services, National Institute of Allergy and Infectious Diseases, 2002:29–35. 11. Milstien J. Regulatory process and three Rs alternatives. In: Brown F, Hendriksen C, Sesardic D, et al. eds. Advancing Science and Elimination of the Use of Laboratory Animals for Development and Control of Vaccines and Hormones. Vol 11. Dev Biol. Basel: Karger, 2002:15–19. 12. Milstien JB. Regulation of vaccines: strengthening the science base. J Public Health Policy 2004; 25(2):173–189.

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13. Anonymous. The PAHO Revolving Fund. EPI Newsl 1979; 1(1):3. 14. Anonymous. EPI revolving fund vaccines prices. EPI Newsl 1983; 5(3):8. 15. Anonymous. Control of vaccines for the EPI program. EPI Newsl1979; 1(2):6. 16. Anonymous. Quality control of biologicals. EPI Newsl 1982; 4 (4):5. 17. Anonymous. The importance of testing. EPI Newsl 1980; II(3):8. 18. WHO Expert Committee on Biological Standardization. Thirtyfirst report. The national control of vaccines and sera. Technical Report Series 1981, 658, Annex 11. 19. WHO Expert Committee on Biological Standardization. Thirtyseventh report. Procedure for Evaluating the Acceptability in Principle of Vaccines Proposed to United Nations Agencies for Use in Immunization Programmes. Technical Report Series 1987, 760, Annex 1. 20. WHO Expert Committee on Biological Standardization. Thirtyeighth report. Modification for lyophilized BCG Vaccines of the Procedure for Evaluating the Acceptability in Principle of Vaccines Proposed to United Nations Agencies for Use in Immunization Programmes. Technical Report Series 1988, 771, Annex 2. 21. WHO Expert Committee on Biological Standardization. Thirty-ninth report. Procedure for Evaluating the Acceptability in Principle of Vaccines Proposed to United Nations Agencies for Use in Immunization Programmes. Technical Report Series 1989, 786, Annex 1. 22. World Health Organization. Immunization, Vaccines and Biologicals. Procedure for Evaluating the Acceptability in Principle of Vaccines Proposed to United Nations Agencies for Use in Immunization Programmes, Document WHO/IVB/05.19. Available at: http://www.who.int/immunization_standards/vaccine_quality/ pq_system/en/index.html. Accessed March 30, 2007. 23. Milstien J, Dellepiane N, Lambert L, et al. Vaccine quality—can a single standard be defined? Vaccine 2001; 2956:1–4.

24. WHO Expert Committee on Biological Standardization, Forty-fifth Report. Regulation and licensing of biological products in countries with newly developing regulatory authorities. Technical Report Series 1995, 858, Annex 1. 25. World Health Organization. Vaccine Supply and Quality. National Control Authority, Guidelines for Assessment of Vaccine Quality in Non-producing Countries, 1995. WHO/VSQ/95.1. 26. World Health Organization. Vaccines and Biologicals. Regulation of Vaccines: Building on Existing Drug Regulatory Authorities, 1999. WHO/V&B/99.10. 27. UNICEF. Vaccines for children: Supply at risk. New York: UNICEF, 2002. 28. World Health Organization. WHO list of vaccines for purchase by UN agencies as of March 2007. Available at: http://www.who. int/immunization_standards/vaccine_quality/pq_suppliers/en/ index.html. Accessed March 30, 2007. 29. WHO. National regulatory authorities (NRAs) and National Control Laboratories (NCLs) in countries producing vaccines prequalified for purchase by UN agencies. Available at: http://www. who.int/immunization_standards/national_regulatory_authorities%20/offices/en/index.html/. Accessed March 30, 2007. 30. Milstien J, Cash RA, Wecker J, et al. The development of priority vaccines for disease-endemic countries: the risk-benefit calculus. Health Aff 2005; 24(3):718–728. 31. EMEA 726/2004. Available at: http://www.emea.europa.eu/htms/ human/noneu_epar/background.htm. Accessed March 30, 2007. 32. Brennan MJ, Fruth U, Milstien J, et al. and the Developing Countries Vaccine Regulatory Network and the Ad Hoc Regulatory and TB Expert Panel. Development of new tuberculosis vaccines: a global perspective on regulatory issues. PLoS Med 2007; 4(8):e252. 33. World Health Assembly Resolution 45.17, mandating vaccine safety and efficacy.

14 Vaccine Safetya Neal A. Halsey Department of International Health, Institute for Vaccine Safety, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A.

Robert Ball Center for Biologics Evaluation and Research, Office of Biostatistics and Epidemiology, Food and Drug Administration, Rockville, Maryland, U.S.A.

Leslie K. Ball Center for Drug Evaluation and Research, Rockville, Maryland, U.S.A.

INTRODUCTION

Injection Related

The benefits of immunization in reducing the morbidity and mortality of infectious diseases are well recognized from both public health and individual perspectives. Widespread use of vaccines in the past century led to eradication of smallpox and control of many infectious diseases including diphtheria, pertussis, measles, and polio (1). These public health triumphs, however, come with the implicit understanding that immunizations are not completely free of risk. Vaccines, like all pharmaceutical products, can have adverse effects ranging from transient common local reactions to rare but serious and irreversible events. Vaccines are held to higher safety standards than drugs because they are usually given to healthy individuals (often children) to prevent an infectious disease rather than to treat an established disease or condition. Also, many vaccines are required for children to attend school in the United States and other countries. Vaccine developers need to construct a comprehensive plan for incorporating vaccine safety throughout the life cycle of the vaccine from preclinical testing through post-marketing. In each phase of the life cycle, different strategies and tools are needed to monitor safety and identify problems as quickly as possible. In this chapter, we review how vaccines cause adverse events, lessons learned from historical events and recent controversies, describe scientific methods for evaluating the safety of vaccines, and review principles of risk management and risk communication for immunization safety.

Injections or fear of injections can cause fainting, which results in injuries, including long-term neurological sequelae from head trauma (2). Injections can also predispose to provocation poliomyelitis because of wild type of live polio vaccine viruses (3). Injections can damage peripheral nerves because of direct trauma or adjacent inflammatory responses (4).

HOW VACCINES CAUSE ADVERSE EVENTS Developers of new vaccines need to evaluate every component of a candidate vaccine for its potential to cause unanticipated and undesirable adverse effects. An understanding of how vaccines can cause adverse events can be helpful for making choices regarding vaccine components and in the development of guidelines for how the vaccine will be stored and administered. How vaccines cause adverse events are given below. a

Contamination Bacterial contamination can occur during handling of vaccines after distribution. Bacterial contamination of multidose diphtheria, tetanus, and pertussis (DTP) vials resulted in several clusters of children with cellulitis, abscesses, or sepsis (5). Multidose vials of measles vaccine have become contaminated with staphylococci and caused sepsis and death when the vials were not discarded at the end of vaccine sessions in India and other countries (6,7). Viral contamination occurred inadvertently during the manufacture of yellow fever vaccines in 1942 when human sera added to yellow fever vaccine resulted in over 50,000 cases of clinical hepatitis in U.S. military personnel with a case fatality rate of 2 to 3 per 1000. Investigation at the time pinpointed the cause as contamination of the human serum used as a stabilizing agent; serological testing decades later confirmed the etiology as hepatitis B (8–10). Some polio vaccines produced prior to 1963 contained SV40 virus, which was not detected earlier because the virus did not cause cytopathogenic effects in the tissue lines that were used for vaccine production. The presence of this virus has not been shown to be associated with any human cancer (11). These experiences highlight the point that infectious agents can be undetectable by current technology and caution is needed with the use of all biological products in vaccine production. Problems in the manufacturing process can occur, including the potential for inadvertent bacterial or fungal contamination. For example, in

The views represented here are those of the authors and not intended to represent those of their respective organizations. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.

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2006 some vials of an influenza vaccine contaminated with Serratia marcescens led to a temporary halt in the production process until new procedures could be put in place to ensure sterility (12). This led to a shortage of influenza vaccines that year in the United States and some other countries. Manufacturing procedures need to be reviewed frequently, and constant quality control vigilance need to be maintained to avoid such problems.

Inflammatory Responses Adjuvants, such as aluminum hydroxide, and other components can stimulate inflammatory responses resulting in local swelling, pain, and tenderness. Lipopolysaccharides and other components in whole-cell pertussis vaccines cause fever, presumably by stimulating the release of inflammatory cytokines after the antigen is ingested by macrophages.

Replication of Live Agents Live measles vaccines cause fever in the 6 to 12 days following vaccination because of replication of the virus. Rubella vaccines can cause arthralgia and arthritis (13), the latter probably by direct infection of the joint by the vaccine virus (14). Oral poliovirus vaccine (OPV) causes paralytic polio in approximately one in every million infants immunized; the mechanism is infection of the anterior horn cells of the spinal cord, the same as for wild-type viruses (15). About one-fourth of the affected individuals have underlying B-cell immune deficiency disorders (16), but most are normal. Investigators have speculated that these individuals have a unique susceptibility to the virus or that, by chance, there was selection of a more neurovirulent virus that is found in very low concentrations in vaccine preparations (15). Smallpox and BCG vaccines cause local infections and, occasionally, more serious complications due to dissemination in patients with immune deficiency disorders (17).

Hypersensitivity Reactions Some vaccines contain allergens including residual media (e.g., egg protein in influenza and yellow fever vaccines) and gelatin used as a stabilizer in measles and varicella vaccines (18). These allergens can induce IgE antibody–mediated immediate hypersensitivity reactions, including urticaria, angioedema, or anaphylaxis (19).

Other Immunologically Mediated Reactions In 1976 influenza vaccines produced in response to an anticipated pandemic of H1N1 influenza were associated with an increased risk of Guillain-Barre´ syndrome (GBS), an autoimmune disorder, in approximately 1 in every 110,000 vaccine recipients (20). No increased risk of GBS following influenza vaccines was noted for many subsequent years. However, in 1993 and 1994, a possible increased risk of GBS was detected: a rate of about one per one million vaccine recipients was noted (21). No other vaccine has been clearly demonstrated to be associated with an increased risk of GBS. However, data suggest a possible association between an increased risk of GBS following quadrivalent meningococcal conjugate vaccine at a rate of approximately 1 per one million vaccinated adolescents (22). One person who developed GBS after meningococcal conjugate vaccine had previously experienced episodes of GBS following DTP and Td. Smallpox vaccine rarely causes

myopericarditis, presumably because of an autoimmune process (23). Other immunologically mediated disorders, such as thrombocytopenia following measles-containing vaccines, occur rarely (24).

Other and Unknown Mechanisms For some adverse events, the mechanism has not been identified. Rhesus rotavirus vaccine (RRV) was associated with an increased risk of intussusception in the three to eight days following vaccination (25). Also, entire-limb swelling following acellular pertussis and other vaccines can occur by unknown mechanisms (26,27). Whole-cell pertussis vaccines were associated with an increased risk of hypotensive-hyporesponsive episodes, and encephalopathy occurred rarely (28). The mechanisms involved in these adverse events have not been clearly identified but are assumed to be caused by direct effects of lipopolysaccharides, toxins, and other components in the pertussis organism stimulating release of host factors that affected various organs. The rates of these disorders are much lower following acellular pertussis vaccines (29). The Vaccine Injury Compensation Program (VICP) has awarded compensation for aggravation of an underlying mitochondrial dysfunction disorder in a girl who developed a regressive encephalopathy following receipt of multiple vaccines at 19 months of age (30). Mitochondrial dysfunction disorders are known to predispose to neurological complications associated with infections and other oxidative stresses (30). Interferon, which is generated in response to viral infections, is known to disrupt some mitochondrial oxidative activity (30). Additional studies are needed to determine the potential role of mitochondrial dysfunction in the pathogenesis of certain adverse events following vaccines, including rare instances of encephalopathy or encephalitis following vaccines that cannot be explained by other causes. There is no complete list of all adverse events caused by vaccines, but the VICP maintains a table of events that are potentially compensable, and more information is available through websites maintained by the U.S. Food and Drug Administration (FDA) (31), the Centers for Disease Control and Prevention (CDC) (32), and the Institute for Vaccine Safety (33). The World Health Organization (WHO) (34) maintains a website on vaccine safety, including a list of websites that meet WHO standards for providing information on vaccine safety.

IMPORTANT SAFETY LESSONS FROM THE HISTORY OF VACCINE DEVELOPMENT Modifications in the regulations for vaccine production and in the guidelines for vaccine use have resulted from lessons learned from real or perceived vaccine safety problems.

Vaccine Purity In St. Louis in 1901, 20 children became ill and 14 died following receipt of an equine diphtheria antitoxin contaminated with tetanus toxin. This event led to the passage of the Biologics Control Act of 1902 (the virus, serum, and toxin law), which included license requirements for biological products and their manufacturing facilities, authority to conduct unannounced inspections, the requirement for accurate product labels, and penalties for noncompliance including revocation of license (35). In 1928 12 children died of staphylococcal sepsis

Chapter 14: Vaccine Safety

in Queensland, Australia following contamination of a multidose container of diphtheria toxin-antitoxin mixture, prompting the investigating committee to recommend that biological products should not be formulated in containers for multiple use unless a sufficient concentration of antiseptic was added to inhibit microbial growth (36). The contamination of yellow fever vaccine with hepatitis B virus and polio vaccines with SV40 virus indicate the need for careful quality control of all materials used in the manufacturing process.

Seed Lots and Standardization of Manufacturing Prior to 1947, yellow fever vaccines were maintained in laboratories and produced as needed in different cell lines on the basis of availability. The demonstration of marked variability of the risk of encephalitis associated with different lots of vaccine in Brazil demonstrated the need for standardizing manufacturing procedures to assure that all lots of live, attenuated viral vaccines have the same characteristics (37,38).

The Cutter Incident In 1955 rapid scale-up of production was required to meet projected demand for inactivated polio vaccine (IPV). Certain lots of vaccine produced by one manufacturer had incomplete inactivation of viruses due to aggregates of poliovirus. Sixty cases of paralytic polio occurred in vaccine recipients, and 89 cases in family contacts in the so-called ‘‘Cutter incident’’ (39,40). This incident highlighted the importance of quality control for every change in the manufacturing process and careful monitoring of vaccine safety post licensure.

Vaccine-Associated Paralytic Polio and Vaccine-Derived Polio Outbreaks In 1964 the Public Health Service commissioned a special advisory committee to evaluate a small number of cases of paralytic polio following receipt of oral polio vaccine (OPV) in vaccinated individuals living in non-epidemic areas. Because no laboratory techniques were available at that time to distinguish vaccines from wild-type polio strains, the committee relied on epidemiological surveillance data between 1962 and 1964. The epidemiological evidence pointed to a causal link with OPV (41). Subsequent developments of laboratory methods, including genetic sequencing, now clearly differentiate wild-type from vaccine-derived polioviruses (42). OPV causes vaccine associated paralytic polio (VAPP) in the recipient or a close contact (43). OPV can also revert to a wild-type phenotype and cause outbreaks of circulating vaccine-derived polioviruses (cVDPV) (44). Also, persistent excretion of polio vaccine viruses for many years can occur in immunodeficient persons and later be transmitted to susceptible persons (44).

Enhancement of Measles and Respiratory Syncytial Virus Disease Following Formalin-Inactivated Vaccines Infant recipients of an investigational formalin-inactivated respiratory syncytial virus (RSV) vaccine developed more severe lower respiratory disease and were more likely to be hospitalized than unvaccinated infants after exposure to RSV many months after vaccination (45,46). Also, some children who received a licensed inactivated measles vaccine developed ‘‘atypical’’ and more severe respiratory disease than unvaccinated children two or more years later when they were exposed

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to wild-type measles virus (47,48). The measles vaccine had been licensed in 1963 on the basis of safety and immunogenicity data as well as demonstrated protective efficacy following exposure to measles in the one year following vaccination. These experiences demonstrated the potential for serious adverse events to take place years after vaccination even with vaccines produced in a manner similar to other successful vaccines without such adverse events (i.e., IPV). These vaccines induced poor-affinity antibody, and protection waned after several months. The enhanced pulmonary disease was caused by antigen-antibody complexes (49). Understanding the pathogenesis of adverse events and development of an animal model open the door to evaluating new candidate vaccines.

Whole-Cell Pertussis Vaccines During the 1970s and 1980s, concerns were raised, first in Japan and Europe and later in the United States, over a possible association between encephalopathy and DTP vaccine (diphtheria and tetanus toxoids and whole-cell pertussis vaccine combination) (50,51). Whole-cell pertussis vaccination was discontinued in Japan and not recommended in Sweden, and some U.S. vaccine manufacturers withdrew from the marketplace, creating the potential for vaccine shortages. A coalition of health professional organizations, consumer advocacy groups, and others pressed for the passage of the National Childhood Vaccine Injury Act of 1986 (52). The Act created the VICP, called for unifying national reporting system for adverse events [the Vaccine Adverse Event Reporting System (VAERS)], mandated comprehensive reviews of vaccine-related adverse events by the Institute of Medicine (IOM), provided for improved record keeping of vaccine administration, and mandated the development and distribution of vaccine information materials. Acellular pertussis vaccines were first developed and marketed in Japan. Comparison studies demonstrated efficacy for several acellular vaccines comparable to or higher than whole-cell vaccines, and the acellular products were associated with reduced rates of fever, febrile seizures, local swelling, tenderness, and pain associated with whole-cell products (53). Acellular products have replaced whole-cell vaccines in most industrialized countries, but highly effective whole-cell vaccines continue to be used in many developing countries, primarily because of the lower cost than that of acellular products. The experience with whole-cell vaccines in industrialized countries highlights the importance of public acceptance of vaccines and the need to make vaccines as safe as possible to maintain public confidence.

Rotavirus Vaccines

RRV (Rotashield 1) was licensed in 1998 on the basis of demonstrated efficacy and safety in several trials. Intussusception was observed during pre-licensure clinical studies in 5 out of 10,054 (0.05%) infants who received RRV and 1 out of 4633 (0.02%) placebo recipients (p > 0.45) (54). Following licensure, reports to VAERS of nine children who developed intussusception after receipt of RRV led to an initial case-control study and a recommendation to temporarily suspend use of the vaccine (55). Case-control/case-series and retrospective cohort studies showed the association between RRV and intussusception to be strong, temporal, and specific, with the attributable risk estimated to be approximately 1 in 5000 to 1 in 11,000 vaccine recipients (25,56). The Advisory Committee on Immunization Practices (ACIP) and the American Academy of

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Pediatrics rescinded the recommendations for RRV in October 1999, and the manufacturer thereupon withdrew the vaccine from the market and ceased production (57,58). The association with intussusception was not detected in the pre-licensure studies because the sample size was inadequate to detect such a rare adverse event (109). Two other manufacturers persevered with the development of other candidate vaccines and conducted multicenter phase III trials with 30,000 to 35,000 vaccine recipients and equivalent numbers of placebo recipients who received vaccine at more restricted early ages (59,60). Neither vaccine was associated with an increased occurrence of intussusception, and both vaccines have been approved for use. This experience highlights the need for conducting trials to address safety questions for some vaccines that are much larger than the size needed to evaluate efficacy. Also, all vaccines of the same type may not have the same safety profile, emphasizing the need for post-licensure studies to identify adverse events that might occur at rates too infrequent to be detected prior to licensure in clinical trials.

Osp A Lyme Disease Vaccine In 1998 a vaccine containing the Osp A protein of Borrelia burgdorferi was licensed on the basis of demonstrated efficacy and no significant increased risk of serious adverse events (61). A similar product developed by another manufacturer was also shown to be efficacious and without serious adverse events (62). Shortly before licensure, studies of patients with treatment-resistant Lyme arthritis indicated a possible cross-reactive amino acid sequence in the Osp A protein with human leukocyte functional antigen (63). Although there was no evidence of increased risk of arthritis following vaccination in either the pre-licensure or the post-licensure safety data, concerns about this possible association undoubtedly tempered the recommendations for use of the vaccine by advisory committees and resulted in limited sales. In addition, class action lawsuits were filed and settled out of court. The manufacturer withdrew the licensure for the vaccine in 2002. Concerns about possible crossreactivity with vaccine components have hindered the development of other vaccines based on components of bacteria that could theoretically trigger autoimmune disorders including group A streptococci (64) and group B meningococci.

FALSE PERCEPTIONS OF SAFETY Concerns about vaccine safety can reduce the acceptance of immunizations with consequent outbreaks of vaccine-preventable diseases even when the scientific data do not support a causal relationship. Alleged links between measles, mumps, and rubella (MMR) vaccine and inflammatory bowel disease and autism were made on the basis of very weak anecdotal observations (65,66). Careful reviews of these hypotheses found them to be without merit (67,68). The concerns, however, resulted in decreased acceptance of MMR in Great Britain and outbreaks of measles (69). In 1999 the U.S. Public Health Service and the American Academy of Pediatrics called for a reduction or removal of thimerosal as a preservative from vaccines administered to infants because the potential cumulative exposure exceeded guidelines for exposure to methylmercury (70,71). After preliminary data suggested the possibility of an association with neurodevelopmental disorders, some groups believed that thimerosal might cause autism (72). Subsequent epidemiological studies and careful evaluation for neurodevelopmental disorders at 7 to 10 years of age have revealed

no consistent evidence of any substantial neurodevelopmental disorder associated with thimerosal exposure (73,74). The complexity of these issues points to the need for a comprehensive and systematic approach to evaluating data on vaccine safety. New issues and concerns arise regularly, indicating the need for ongoing programs to address safety concerns.

VACCINE SAFETY ACROSS THE LIFE CYCLE At the earliest stages of development, vaccine researchers should develop a comprehensive plan to acquire necessary data on safety and efficacy during all phases of development. Numerous judgments will be required as to the type and quantity of safety data required to support licensure, including preclinical toxicity testing and sample size of clinical studies. The required data will differ for a vaccine intended for universal use in children as compared with one to be used in more niche populations such as adult travelers. Depending on the product class, some potential safety concerns are known a priori or can be predicted from the historical experience of development of similar vaccines. Understanding the background incidence of illness occurring in the target population and anticipating risks that might be perceived as related to vaccination may be as important as understanding the product-related risks, because ultimately the intended population will need to be convinced that the vaccine is safe enough to use. The developer can begin this process by identifying the diseases in the target population that might be suspected to be related to the vaccine because onset or diagnosis may coincide with the timing of immunizations. Neurological, rheumatological, and other immune-mediated disorders have been falsely attributed to vaccines when other etiologies could not be established. In addition, disorders that are not well understood but have occurred with some temporal relation to immunization, such as sudden infant death syndrome or Gulf War syndrome, have also been linked with immunization in the minds of the public even though the scientific data do not support a causal relationship. Developers should decide whether it is necessary to develop evidence that the vaccine does not cause a particular condition when such perceptions may affect public acceptance of the vaccine. Ultimately, safety assessments should provide adequate data to support licensure and recommendations for use as well as communicating the benefits and risks of the vaccines. Responsibility for evaluating vaccine safety begins with vaccine researchers and/or manufacturers and later extends to regulatory authorities, other government and international agencies, immunization advisory bodies, and health care providers. Much attention has been devoted to immunization safety efforts in the last decade in the United States and worldwide. The WHO has made immunization safety a global priority (75,76). The International Conference on Harmonisation (ICH), a collaborative effort of regulatory authorities and pharmaceutical companies in Europe, Japan, and the United States to harmonize requirements for registering pharmaceutical products, has developed recommendations on a range of preclinical, clinical, and post-licensure safety evaluations (77). Safety, as defined by the FDA, is ‘‘the relative freedom from harmful effect to persons affected directly or indirectly by a product when prudently administered, taking into consideration the character of the product in relation to the condition of the recipient at the time’’ (78). Thus, safety is relative and relational; it depends on the benefit/risk assessment at a particular point in time, the specific indication, and the intended recipient.

Chapter 14: Vaccine Safety

Preclinical Testing The diversity of biological products necessitates individualization of the preclinical evaluation, however, certain general principles apply. Prior to introduction of an investigational vaccine in human volunteers, investigators are required to provide evidence supporting the scientific rationale for a vaccine candidate, including immunogenicity, the quality (including purity) of the product, and preclinical safety information. Animal models are useful for evaluating disease pathogenesis, immune response, toxicity, and, in some cases, efficacy against challenge with the infectious disease that the vaccine is intended to prevent. Animal studies are usually overseen by independent committees, often called animal use committees, to assure the humane conduct of studies and to avoid unnecessary testing and sacrifice of animals. Product quality is assessed by evaluating the manufacturing process, the materials used during production, and the final product. Specific descriptions of the manufacturing process, documentation of the source and quality of the materials used in manufacture, and in-process testing help to characterize the safety of the product (79). In addition, ICH has developed a wide range of guidance documents on quality assurance and in vitro and in vivo preclinical studies (77). Concerns about transmissible spongiform encephalopathies have highlighted the need to document the sources of bovine-derived materials and have led the United States to exclude materials for vaccine manufacture from countries in which bovine spongiform encephalopathy (BSE) or BSE risk exists (80–82). Careful attention should be given to the design of preclinical toxicity studies, particularly when the investigational product consists of components not previously studied in humans such as new antigen delivery systems and novel adjuvants (83). Vaccines intended for administration to pregnant women or women of childbearing potential should be evaluated for teratogenicity and developmental toxicity (usually in several animal species) (84). Additional laboratory testing may be warranted to evaluate particular products, for example, adventitious agent testing for vaccines produced in animal or human cell substrates (85), preclinical studies evaluating the potential for integration of plasmid DNA into the host genome for DNA vaccines (86,87), and demonstration of adequate attenuation for live, attenuated vaccines (88). Testing is required to document sterility, general safety, potency, and purity of the vaccine (89). Extra vigilance is necessary for novel vaccine technologies that require the development and standardization of new quality control measures. Laboratory evaluation of vaccine safety does not end when clinical studies begin or when a vaccine is licensed. Changes in the manufacturing process or components, the development of enhanced testing techniques, as well as new safety concerns identified in clinical studies or in post-marketing surveillance of a vaccine may prompt the investigator and/or manufacturer to consider reevaluation of the product’s preclinical safety.

Clinical Pre-Licensure Studies Clinical Studies and Human Subject Protections Clinical studies must comply with accepted ethical principles guiding human participation in clinical trials, including informed consent, equitable selection of subjects, and appropriate scientific and ethical review of the proposed study. Evolution in thought regarding elements of ethical research,

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as well as the use of new vaccine technologies with uncertain risks, present new challenges for ensuring participant safety in clinical trials. Human subject protections are guided by ethical principles formalized in consensus documents such as the Belmont Report (90), various iterations of the Declaration of Helsinki (World Medical Association), and the International Ethical Guidelines for Biomedical Research Involving Human Subjects (91). Similar concepts are codified in U.S. Department of Health and Human Services (DHHS) regulations as the ‘‘Common Rule’’ (92) adopted by 17 federal agencies that support or conduct research with human subjects, and FDA regulations that govern drug, biological, and device research (93). Centers that conduct human trials are required to have institutional review boards (IRBs), which independently review and approve the studies prior to any human testing. The IRBs also monitor the safety of human subjects during clinical trials. Investigators are usually required to have independent data monitoring committees (DMCs), also known as data and safety monitoring boards (DSMBs), to evaluate adverse events during the course of trials. DMCs or DSMBs consist of individuals with relevant expertise who provide ongoing review of data accumulated during clinical studies. Members of DMCs or DSMBs should be independent of the investigators and the study sponsors, and have no conflicts of interest with the product to be evaluated. Small, phase I, open studies may often have only a medical monitor to assess adverse events. The role of a DMC or DSMB is to advise the investigators and the sponsors on the safety of current study participants and the continuing validity and scientific merit of the study (94). A DMC or DSMB should have the ability to unmask the participants by a study group at any point during clinical trials to evaluate potential safety concerns. Draft guidance is available from the FDA to help determine when a DMC is needed and how such committees should operate. Ethical and practical issues facing investigators with respect to pivotal studies include the choice of research design, the use of placebo controls, or the use of a comparison vaccine when evaluating a vaccine against a disease for which a licensed vaccine already exists (95,96). Conducting clinical studies in international settings presents additional challenges such as ensuring adequate local review and oversight, the need for studies to be relevant to the health needs of the host country, and the sustaining newly introduced health interventions once the trial is completed (97–99). Challenge studies to valuate vaccine efficacy, that is, inducing clinical infection in subjects to study the efficacy of an experimental vaccine, presents ethical issues for subject safety (100,101). When planning challenge studies, investigators should carefully consider the seriousness of the infectious disease, including sequelae, and the ability to treat the infection. Demonstrating efficacy when field efficacy trials or human challenge studies are not feasible or are unethical, for example, vaccines against agents of bioterrorism, is problematic. For products that reduce or prevent serious or life-threatening conditions where the product is expected to provide meaningful benefit over existing approaches, the FDA has regulations (the ‘‘animal rule’’) describing how animal efficacy data can be used to support licensure (102). In this setting, human clinical safety and immunogenicity data would still be required. The approval of new vaccines against anthrax and smallpox is an example of the development of new vaccines against agents where it is not possible to assess efficacy through field trials or individual challenge studies (103,104).

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Phases of Clinical Studies Human clinical studies are allocated into phases for regulatory purposes (105). In the United States, when a new vaccine is first tested in humans, a sponsor (a vaccine manufacturer, academic investigator, government agency, or other individual or organization) must first submit an investigational new drug (IND) application to the FDA or some other regulatory agency (106). During each phase, clinical studies should be designed and conducted under conditions that optimize human subject protections and provide sufficient data to proceed to the next phase. If data at any stage of clinical development raise significant concerns regarding safety, the regulatory authority may request additional information or may halt ongoing or planned studies through a ‘‘clinical hold’’ (107). Phase I clinical trials, the initial testing in humans of safety and immunogenicity, generally involve between 10 and 100 subjects. These studies often include limited dose ranging to provide preliminary information on immune responses and the corresponding safety profile at particular doses. Adverse event monitoring is based on previous experience with the investigational product and related products, but careful daily evaluations are usually required. The use of clinical criteria defined a priori for halting further administration to subjects (i.e., ‘‘stopping rules’’) may be considered. If vaccines are ultimately intended for infants, a stepwise approach is often used with initial studies conducted in adults and older children before testing in infants. Phase II studies often include up to several hundred individuals to evaluate safety and immunogenicity and usually include blinding, randomization, and controls. Assessments often include simultaneous administration with other immunizations routinely administered at the same time to address the possibility of altered immune responses or increased risk of side effects, such as fever. These studies can evaluate only the most common types of adverse events. Phase III studies evaluate efficacy and safety and are usually the pivotal trials for licensure. These studies are almost always randomized and controlled. The sample size is usually determined by the number required to establish efficacy and may range from a few hundred to tens of thousands of subjects. The sample sizes for pivotal immunogenicity studies may be much lower than what would be required to demonstrate clinical efficacy. Phase III vaccine studies usually have limited ability to detect rare adverse events (Table 1). To detect a doubling of less common adverse events, such as those occurring at a background rate of 1 in 100, requires approximately 5000 subjects. Clinical trials involving 50,000 individuals would be needed to detect doubling of an adverse event with a Table 1 Sample Sizes Needed to Detect Increased Rates of Rare Adverse Events After Immunization Rates (%) 1.0 vs. 2.0 1.0 vs. 3.0 0.1 vs. 0.2 0.1 vs. 0.3 0.05 vs. 0.1 0.01 vs. 0.02 0.01 vs. 0.03

Sample sizea

No. potentially affected per million vaccine recipients

5000 1750 50,000 17,500 100,000 500,000 175,000

a Two-arm trial, power = 80%, a (two sided) = 5%. Source: Adapted from Refs. 108 and 109.

10,000 20,000 1000 2000 500 100 200

background incidence of 1 in 1000 (108,109). Some experts have advocated the use of expanded ‘‘simple’’ trials prior to licensure to provide more precise data on risks of uncommon adverse events (109). The safety database prior to licensure may include a relatively smaller number of vaccine recipients (*10,000) compared with the number of recipients who might receive the vaccine (e.g., *4,000,000 infants in U.S. birth cohort per year), so an increased risk of adverse effects occurring at a rate of 1 in 1000 or lower is unlikely to be identified. Also, clinical trials often exclude subgroups of the general population such as the immunocompromised, preterm infants and individuals with chronic or self-limited illnesses, and may have limited data from different racial or ethnic groups or geographic locations. Thus, pre-licensure clinical studies may not address the variation in susceptibility to adverse effects that exists in the general population.

Vaccine Licensure In the United States, the sponsor and the FDA present their findings to the Vaccines and Related Biological Products Advisory Committee (VRBPAC), an external committee of experts, in an open public meeting for comment and advice on interpretation of the submitted data and other issues related to the acceptability of the new vaccine. The approval process also entails the provision of adequate information to health care providers and the public in the form of a product label that describes the vaccine’s proper use, including its potential benefits and risks, and any contraindications.

Post-Licensure Assessment of Vaccine Safety The FDA requests vaccine manufacturers to submit a pharmacovigilance plan, commonly called a phase IV study, with their biologics license application (BLA) (110). This ensures adequate time for review of these plans with the review of other safety data submitted with the BLA. A detailed pharmacovigilance plan is important to ensure that adequate safety monitoring will be conducted post licensure. A pharmacovigilance plan should provide a summary of ongoing safety issues (e.g., important identified risks, potential risks, and/or missing information), outline routine pharmacovigilance practices (systems and processes for collecting and reporting adverse events, preparation of reports for regulatory authorities, continuous monitoring of the safety profile, and other requirements deemed necessary), an action plan for safety issues, and a summary of actions to be completed including milestones (111). Additional clinical studies agreed to by the sponsor as a condition of licensure may include active and passive surveillance for unexpected adverse events after licensure, and targeted clinical, epidemiological, and laboratory studies to evaluate safety concerns. Phase IV studies usually involve active surveillance in defined populations to identify rare events that could not be detected prior to licensure. The use of automated databases, such as those administered by health maintenance organizations, facilitates collection of data on less common adverse events. Detailed information on common local and systemic adverse events may be collected in a subset of individuals using diary cards or telephone or clinic followup. These studies typically take place in the first few years after licensure, with information expected to be presented in a timely fashion to regulatory agencies and immunization advisory groups.

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Passive Surveillance Systems Post-marketing surveillance involves monitoring reports of adverse events to vaccine manufacturers or regulatory authorities followed by evaluation of possible ‘‘signals’’ of problems that had not been previously identified. In the United States, surveillance of adverse events after vaccination is undertaken using the VAERS, which is jointly managed by the FDA and CDC (112,113). VAERS receives over 15,000 adverse event reports annually; approximately 10% to 15% are reported as serious, defined as life threatening, requiring hospitalization or prolongation of hospitalization, a persistent or significant disability/incapacity or a congenital anomaly/birth defect or a medical event that may require intervention to prevent one of the above situations (114). Information on VAERS is available on the Internet (31). The WHO recommends that all countries should have national systems to report and evaluate adverse events (115). WHO has established the Global Advisory Committee on Vaccine Safety to make independent assessments of vaccine safety issues (34,75). Data from many countries are aggregated by the WHO Collaborating Centre for International Drug Monitoring at Uppsala, Sweden, including some data from VAERS. This center has developed a data-mining technique for routine monitoring of this database and routinely publicizes possible signals. The United Kingdom’s Medicine Control Agency (MCA) enhances their surveillance for new products with the ‘‘yellow card’’ system. New products are distributed with a reporting form (the ‘‘yellow card’’) to highlight the need for clinicians and pharmacists to be alert for and report adverse events. If an adverse event occurs, the reporter sends the form to the MCA and the Committee on Safety of Medicine so that the adverse event can be evaluated. Passive surveillance systems are useful for detecting unrecognized adverse events, monitoring known reactions, identifying possible risk factors, and vaccine lot surveillance (112,113,116). Priorities in analyzing VAERS data include adverse events reported after recently licensed vaccines, issues that have been identified to be of particular concern to the public, and rare adverse events not likely to be identified in clinical trials or controlled post-marketing safety studies [e.g., intussusceptions (117) and Stevens–Johnson syndrome (118)]. Special studies triggered by VAERS reports might include revisions of the range of clinical signs and symptoms, investigation of potential risk factors, and pathophysiology [e.g., thrombocytopenia (119), syncope (120), hypotonic-hyporesponsive episodes (HHEs) in infants (121), and neurological complications following yellow fever vaccine (122)]. All reports of serious adverse events (including death) following vaccination are reviewed by FDA medical officers as they are received. Periodically, vaccine-specific surveillance summaries are prepared to describe reported adverse events and to look for unexpected patterns in clinical conditions that might suggest a causal link between the vaccine and the clinical condition (28,123–126). Limitations of surveillance systems include lack of verification of reported diagnoses, lack of consistent diagnostic criteria for all cases with a given diagnosis, wide range in data quality, underreporting, inadequate denominator data, and lack of an unvaccinated control group (112,113,127,128). The validity of reported diagnoses and completeness of information in VAERS reports have only been formally studied for a few conditions. In one study, between 26% and 51% of reports of encephalopathy, encephalitis, and multiple sclerosis lacked sufficient information to make a diagnosis (129). Enhanced

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follow-up is sometimes conducted to systematically collect information to evaluate signals (118,121,129). Development of standardized case definitions has been of value for evaluations of adverse events reported to VAERS (129,130). The Brighton Collaboration, an international effort to develop standardized case definitions of adverse events following vaccination, has developed 24 case definitions (131–134) that can be used as guidelines for evaluation of reports in formal reporting systems or in clinical trials. Evaluation of signals usually requires epidemiological methods, sometimes combined with clinical and laboratory analysis. CDC established the Vaccine Safety Datalink (VSD) as a resource for conducting cohort studies using large administrative databases maintained by health maintenance organizations to evaluate specific hypotheses (135,136). VSD studies have found increased risks of intussusception after rotavirus vaccine (56), febrile but not afebrile seizures after DTP or MMR vaccines (137), thrombocytopenia following MMR (24), and apnea following vaccines in premature infants (138). VSD studies have also provided important data regarding the lack of associations between adverse events and vaccines. For example, no difference was found in adverse events by brand of hepatitis B vaccine (139), and no association was found between rubella vaccine and chronic arthropathy in women (140), childhood vaccination and type 1 diabetes (141), and measles-containing vaccines and inflammatory bowel disease (142). Other population-based databases to study vaccine safety concerns include the General Practice Research Database (GPRD) in the United Kingdom (143). Recent examples of the use of the GPRD include studies showing no association between MMR vaccine and increased incidence of autism (144), OPV and intussusception (145), and inactivated influenza vaccine and Bell’s palsy (146). Ad hoc studies are sometimes needed to study rare adverse events if the vaccine or adverse event of concern is not sufficiently represented in databases such as the VSD or if confirmation of a study outcome is sought in a different population. For example, no increased risk of multiple sclerosis or exacerbation of multiple sclerosis following hepatitis B vaccine was found using the Nurses Health Study database and the European Database for Multiple Sclerosis (147–149). A separate study in France where the concern first arose has shown no association between hepatitis B vaccine and childhood onset of multiple sclerosis (150). Similarly, a special study was conducted to estimate an excess risk of one case of GBS per million people administered influenza vaccine in the 1992–1993 and 1993–1994 flu seasons (151). Structured clinical and laboratory evaluations of individual cases can provide valuable data on other causes for adverse events, risk factors, and possible pathophysiological mechanisms. To provide a systematic resource for case-based evaluations, the CDC has organized the clinical immunization safety assessment (CISA) (152). These centers serve as referral centers for clinical vaccine safety questions, develop clinical protocols for the evaluation and management of adverse events possibly related to immunization, systematically evaluate patients with similar adverse events to identify mechanisms of action and risk factors, and develop and test protocols for revaccination of people who have experienced adverse events, as has been done recently in Australia (153). CISA studies have demonstrated no association between SV40 virus infections that might have come from polio vaccines

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and non-Hodgkin’s lymphoma (154) and evaluated risk factors for entire-limb swelling following diphtheria, tetanus, and acellular pertussis (DtaP) vaccines (155). Genetic studies have the potential for identifying specific markers for increased risk of common or rare adverse events. Higher frequencies of specific HLA loci were found in women who developed joint symptoms following rubella vaccination as compared with placebo recipients with joint symptoms (156), and autoimmune encephalomyelitis following Semple rabies vaccine (157). Wilson et al. identified potential genes associated with myopericarditis following smallpox vaccine (36). Specific gene associations with the development of fever following smallpox vaccine have been found (158). Other studies to evaluate polymorphisms in other genes influencing immune and inflammatory responses, such as T-cell receptors and cytokines, might prove to be useful in assessing adverse events (157,159,160). Research in this area could potentially help identify individuals at risk of serious adverse events who could be excluded from receiving selected immunizations and possibly modify vaccines to reduce the risk of rare adverse effects.

CAUSALITY ASSESSMENT Criteria for Causality General considerations for causality assessment, patterned after criteria established by Hill in 1965 (161) and modified by others (162,163), have been generally accepted in the field of epidemiology for causal inference. WHO encourages the use of these criteria (7). l

l

l

l

l

l

Strength of association: The strength of an association refers to the magnitude of the measure of effect of an exposure, usually the relative risk or odds ratio, in a study comparing an exposed and an unexposed group. The larger the magnitude of the effect, the less likely any observed effect is due to chance, bias, or confounding (164,165). Ecological studies alone are not generally accepted as strong evidence of causality, because they do not link individual exposure to individual outcome and can be subject to confounding by unknown or uncontrollable factors. Dose-response relation: A dose-response relation is defined as an increased strength of association with increased magnitude of exposure. Temporally correct association: Exposure must precede the event. This consideration may be limited by the lack of knowledge of the pathogenesis and natural history of an adverse event. Consistency of association: An association should be reproducible in a variety of studies, using different study populations, investigators, and study methods. Specificity of an association: Uniqueness of an association between an exposure and an outcome provides a stronger justification for a causal interpretation than that when the association is nonspecific. However, perfect specificity between an exposure and an effect cannot be expected in all cases because of the multifactorial etiology of many disorders. Many rare adverse events occurring in temporal association with vaccines often have multiple etiologies. Biological plausibility: The existence of a possible mechanism of action that fits the existing biological or medical knowledge is helpful when assessing causality. However, it is easy to generate a theory as to how a vaccine might cause an effect. Biological plausibility is helpful, but not

necessary, criterion for establishing a causal relationship. Causal associations have been found where there was no prior knowledge of biological plausibility. Hill also included ‘‘experimental evidence,’’ ‘‘analogy,’’ and ‘‘coherence’’ as additional considerations in his original discussion of causal inference (161,162). Experimental evidence from clinical trials is seldom available for rare adverse events. ‘‘Analogy’’ has not been accepted as a strong evidence of causality because analogies between exposure and a particular condition can often be drawn, even when causal relationships do not exist. Coherence ‘‘implies that a cause-and-effect interpretation for an association does not conflict with what is known of the natural history and biology of the disease’’ (162). This guideline is similar to biological plausibility, but it might help when comparing the strength of considerations supporting a causal association with the strength of alternative explanations. Evidence for a causal association usually includes an increased risk of the adverse event in vaccine recipients as compared with controls or specific evidence of a vaccine component in affected tissues at a time when it would not be expected with supporting pathological changes and other evidences (166). Examples of evidence for a vaccine component in affected tissues include yellow fever vaccine–associated viscerotopic syndrome (167) and identification of measles vaccine virus in the lung tissue of a patient with AIDS (168). Caution is indicated to rule out the possibility of coincidental infections by wild-type virus that might be interpreted as caused by the vaccine, for example, children who developed chicken pox less than two weeks after receipt of varicella vaccine. Genetic sequencing of the viruses from these children usually revealed wild-type virus; the children had undoubtedly been exposed to chicken pox in the community prior to the admission of the vaccine (169). Exceptions to the general rules stated above include local adverse events at the site of injections and immediate hypersensitivity reactions. Local reactions are almost always caused by material injected at that site, but trauma or a preexisting lesion at the site could cause confusion. Immediate hypersensitivity reactions that occur within minutes of receipt of a vaccine cannot usually be explained by exposure to other allergens. The pathogenesis involves release of histamine from mast cells due to preexisting IgE-specific antibody against a vaccine component. Immediate hypersensitivity reactions can occur up to several hours after a vaccine is administered, and exposures to other potential antigens, such as foods, should be ruled out. A complementary approach to evaluating vaccine safety concerns is the use of formal risk assessment methods, patterned after methods used for environmental exposures. The National Academy of Sciences approach involves hazard identification, dose-response analysis, exposure assessment, and risk characterization (170). Complete information on human exposure and outcomes is often unavailable, so risk assessments have sometimes extrapolated outcomes in animals to humans and from high-dose to low-dose exposures. In such scenarios, risk assessment serves a more qualitative purpose of identifying data gaps and framing the range of possible risk, to aid with policy-making and prioritization of research. Risk assessment methods have been applied to evaluate the possible effects of thimerosal in vaccines (68,71,171), as well as the risk of BSE from using bovine-derived products in vaccine manufacture (81). The risk assessment of thimerosal was primarily qualitative and identified important gaps in

Chapter 14: Vaccine Safety

knowledge. In contrast, the risk assessment of BSE from vaccines resulted in a quantitative estimate of the risk that was extremely low. Risk assessment is likely to be used more often as mechanistic knowledge of adverse event pathophysiology improves and can be mathematically modeled. The limitations of data available in passive surveillance systems usually preclude the assessment of causal assessments for individual case reports. Nevertheless, some expert groups have reviewed individual cases for causality assessment using Bayesian probability (172) and/or a standardized algorithm such as the procedures used in Canada (173). The latter method was adapted by the Anthrax Vaccine Expert Committee to evaluate reports of adverse events following anthrax vaccine for the Department of Defense and for smallpox vaccination in the civilian and military populations (4,174). This approach involves systematic review of individual cases to make a diagnosis, search for known etiologies, and assess the biological plausibility of causal relationships using expert opinion. Similar approaches have been considered for use in drug adverse event causality assessment, but the use has remained controversial when applied to unexpected adverse events (175–177). Analyses of VAERS data usually focus on describing clinical and demographic characteristics of reports and looking for ‘‘signals’’ of adverse events plausibly linked to a vaccine that can be explored through epidemiological studies (125,178). Several quantitative approaches have been proposed that involve identifying conditions reported more commonly after a certain vaccine than others administered to similar populations. These methods are often referred to as ‘‘data mining’’ (142,143). Signals generated through such quantitative analysis are usually subject to clinical and descriptive epidemiological analyses. These semiautomated approaches help improve the efficiency of screening tens of thousands of adverse events reported annually (179), although their utility in replacing traditional case-series evaluations remains to be seen (180,181).

Institute of Medicine Safety Review Committee Reviews Recognizing the continued need for independent comprehensive analyses of vaccine safety, the CDC and National Institutes of Health (NIH) asked the IOM to establish an independent immunization safety review committee. The committee reviewed immunization safety issues by examining the current biological and epidemiological evidences of causality, the biological mechanisms of adverse events, and the larger societal context. The committee has released reports on MMR and autism (72), thimerosal-containing vaccines and neurodevelopmental disorders (73), multiple immunizations and immune dysfunction (182), hepatitis B vaccine and neurological disorders (183), and MMR and thimerosal and autism (72). A separate committee to evaluate the safety and efficacy of anthrax vaccine was convened at the request of the Department of Defense (176,184,185). Some researchers have suggested establishing an independent national vaccine safety board to provide an ongoing, independent oversight of vaccine safety investigations that might help maintain public confidence in vaccines and vaccine programs (186).

RISK MANAGEMENT AND COMMUNICATION Risk management is the process of maximizing the benefits of vaccines while minimizing associated risks (187). Participants in formal risk management decisions include scientists

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developing vaccines, manufacturers, regulatory agencies, and immunization advisory bodies including the Advisory Committee on Immunization Practices for CDC, the Committee on Infectious Diseases for American Academy of Pediatrics, and the Strategic Advisory Group of Experts (SAGE) for WHO. Health care providers, patients, and legislators also make risk management decisions. Recognizing that vaccines can, on rare occasions, cause serious adverse effects and providing compensation to those who develop serious complications following vaccines through the VICP have been important aspects of risk management in the United States in recent years. Successful immunization strategies require an understanding of the ethical underpinnings of immunization policy (188), the role of risk perception in vaccination decisions (165,189), and optimal risk communication strategies (190,191). At the heart of ethical immunization policies is the equitable allocation of benefits and risks between individuals and society. A systematic approach for developing immunization policy and integrating epidemiological, economic, and ethical concerns may help focus risk management decisions and sustain societal consensus on immunizations (188). Factors influencing how likely individuals are to accept risks include whether risks are voluntary and controllable, natural (as opposed to man made), and memorable (192). Risk communication research has demonstrated that individuals are unlikely to undertake a risk control measure, such as immunization, unless they perceive both a serious threat and some control over it (193), emphasizing the importance of involving individuals in immunization decisions. In contemporary society, individuals seek and receive immunization information from a variety of sources, which are not always reliable. The Internet not only provides a wealth of relevant information but also provides unfiltered anecdotes and misinformation regarding adverse events following vaccination (194). The media play a critical role in shaping public perceptions on vaccine benefits and risks. Dissemination of accurate and meaningful information on the benefits and risks of immunization is essential to maintain public confidence; misinformation can adversely impact informed decision-making on the individual and the community levels (195,196). Publicity regarding safety concerns for vaccines administered to infants can make it very difficult for parents to determine who to believe and what information to trust, resulting in immunization delays or refusal (188,197,198). Also, of importance are discrepancies between how scientists explain health risks and what the public believes (199). The development and dissemination of accurate and balanced information at the time of vaccine licensure, and the prompt evaluation of new safety concerns are essential to maintaining public trust in immunization programs (200).

CONCLUSION The current system of vaccine development and monitoring has resulted in vaccines with very low risks of serious adverse effects. Nevertheless, new averse events caused by vaccines are being identified, and the development of new vaccines brings challenges with regard to the potential for real and perceived vaccine safety issues. Continued vigilance regarding immunization safety at all stages of product development is necessary to ensure the safety of new vaccines and to maintain public confidence in vaccines. The challenge remains to apply the best scientific methods to the task and effectively communicate the science to the public.

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ACKNOWLEDGMENTS We are grateful for the comments of Susan S. Ellenberg, PhD, Miles M. Braun, MD and MPH, Karen Goldenthal, MD, Norman Baylor, PhD, and the editorial assistance of Jan Kelliher and Tina Proveaux.

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152. Centers for Disease Control and Prevention. Notices—grants and cooperative agreements: Clinical Immunization Safety and Assessment Centers. Fed Reg 2001. Available at: www.cdc.gov/ vaccinesafety/cisa. 153. Gold M, Goodwin H, Botham S, et al. Re-vaccination of 421 children with a past history of an adverse vaccine reaction in a special immunisation service. Arch Dis Child 2000; 83:128–131. 154. Rollison DE, Helzlsouer KJ, Halsey NA, et al. Markers of past infection with simian virus 40 (SV40) and risk of incident nonHodgkin lymphoma in a Maryland cohort. Cancer Epidemiol Biomarkers Prev 2005; 14:1448–1452. 155. Rennels MB, Black S, Woo EJ, et al. Safety of a fifth dose of diphtheria and tetanus toxoid and acellular pertussis vaccine in children experiencing extensive, local reactions to the fourth dose. Pediatr Infect Dis J 2008 [Epub ahead of print]. 156. Mitchell LA, Tingle AJ, MacWilliam L, et al. HLA-DR class II associations with rubella vaccine-induced joint manifestations. J Infect Dis 1998; 177:5–12. 157. Piyasirisilp S, Schmeckpeper BJ, Chandanayingyong D, et al. Association of HLA and T-cell receptor gene polymorphisms with Semple rabies vaccine-induced autoimmune encephalomyelitis. Ann Neurol 1999; 45:595–600. 158. Stanley SL Jr, Frey SE, Taillon-Miller P, et al. The immunogenetics of smallpox vaccination. J Infect Dis 2007; 196(2):212–219. 159. Hacker UT, Jelinek T, Erhardt S, et al. In vivo synthesis of tumor necrosis factor alpha in healthy humans after live yellow fever vaccination. J Infect Dis 1998; 177:774–778. 160. Pourcyrous M, Korones SB, Crouse D, et al. Interleukin – 6, C-reactive protein, and abnormal cardiorespiratory responses to immunization in premature infants. Pediatrics 1998; 101:e3. 161. Hill AB. The environment and disease: association or causation? Proc R Soc Med 1965; 58:295–300. 162. Rothman KJ. Modern epidemiology. Boston: Little Brown & Company, 1986:23–34. 163. Rothman KJ, Greenland S. Causation and causal inference in epidemiology. Am J Public Health 2005; 95(suppl 1):S144–S150. 164. Wydner EL. Epidemiological issues in weak associations. Int J Epidemiol 1990; 19:S5–S7. 165. Monson RR. The interpretation of epidemiological data, in ‘‘Occupational Epidemiology.’’ Boca Raton, FL: CRC Press, 1980. 166. Halsey NA. The science of evaluation of adverse events associated with vaccination. Semin Pediatr Infect Dis 2002; 13:205–214. 167. Belsher JL, Gay P, Brinton M, et al. Fatal multiorgan failure due to yellow fever vaccine-associated viscerotropic disease. Vaccine 2007; 25:8480–8485. 168. Angel JB, Walpita P, Lerch RA, et al. Vaccine-associated measles pneumonitis in an adult with AIDS. Ann Intern Med 1998; 129:104–106. 169. Sharrar RG, LaRussa P, Galea SA, et al. The postmarketing safety profile of varicella vaccine. Vaccine 2000; 19:916–923. 170. National Research Council, Committee on the Institutional Means for Assessment of the Risks to Public Health. Risk Assessment in the Federal Government: Managing the Process. Washington, DC: National Academy Press, 1983. 171. Clements CJ, Ball LK, Ball R, et al. Thiomersal in vaccines (letter). Lancet 2000; 355:1279–1280. 172. Fenichel GM, Lane DA, Livengood JR, et al. Adverse events following immunization: assessing probability of causation. Pediatr Neurol 1989; 5:287–290. 173. Collet JP, MacDonald N, Cashman N, et al. Monitoring signals for vaccine safety: the assessment of individual adverse event reports by an expert advisory committee. Bull World Health Org 2000; 78:178–185. 174. Neff J, Modlin J, Birkhead GS, et al. Monitoring the safety of a smallpox vaccination program in the United States: report of the joint Smallpox Vaccine Safety Working Group of the advisory committee on immunization practices and the Armed Forces Epidemiological Board. Clin Infect Dis 2008; 46(suppl 3): S258–S270.

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175. Halsey NA. Anthrax vaccine and causality assessment from individual case reports. Pharmacoepidemiol and Drug Saf 2002; 11:185–187. 176. Sever JL, Brenner AI, Gale AD, et al. Response to editorial by Dr. Neal Halsey. Pharmacoepidemiol Drug Saf 2002; 11:189–202. 177. Jones JK. Determining causation form case reports. In: Strom BL, ed. Pharmacoepidemiology. 3rd ed. New York, NY: John Wiley and Sons, 2000. 178. Mortimer E. Stevens Johnson syndrome after vaccination. Pediatr Infect Dis J 2001; 20:818–819. 179. Bortnichak EA, Wise RP, Salive ME, et al. Proactive safety surveillance. Pharmacoepidemiol Drug Saf 2001; 10:191–196. 180. Banks D, Woo EJ, Burwen DR, et al. Comparing data mining methods on the VAERS database. Pharmacoepidemiol Drug Saf 2005; 14:601–609. 181. Almenoff J, Tonning JM, Gould AL, et al. Perspectives on the use of data mining in pharmacovigilance. Drug Safety 2005; 28:981–1007. 182. Stratton K, Wilson CB, McCormick MC, eds. Immunization safety review: multiple immunizations and immune dysfunction. Washington, DC: National Academy Press, 2002. 183. Stratton K, McCormick M, eds. Immunization safety review: Hepatitis B vaccine and demyelinating neurological disorders. Washington, DC: National Academy Press, 2002. 184. Joellenbeck LM, Zwanziger L, Durch JS, et al., eds. The anthrax vaccine: is it safe? Does it work? Report from the institute of medicine. Washington, DC: National Academy Press, 2002. 185. Payne DC, Franzke LH, Stehr-Green PA, et al. Development of the vaccine analytic unit’s research agenda for investigating potential adverse events associated with anthrax vaccine adsorbed. Pharmacoepidemiol Drug Saf 2007; 16(1):46–54. 186. Salmon DA, Moulton LH, Halsey NA. Enhancing public confidence in vaccines through independent oversight of postlicensure vaccine safety. Am J Public Health 2004; 94:947–950. 187. Food and Drug Administration. Managing the risks from medical product use: creating a risk management framework. Report to the FDA Commissioner from the Task Force on Risk Management. May 1999. Available at: www.fda.gov/oc/tfrm/riskmanagement.html. Accessed April 14, 2008.

188. Feudtner C, Marcuse EK. Ethics and immunization policy: promoting dialogue to sustain consensus. Pediatrics 2001; 107: 1158–1164. 189. Gellin BG, Maibach EW, Marcuse EK. Do parents understand immunizations? A national telephone survey. Pediatrics 2000; 106:1097–1102. 190. Evans G, Bostrom A, Johnston RB, et al. Risk communication and vaccination, workshop summary, Institute of Medicine, Washington, DC: National Academy Press, 1997. 191. Ball LK, Evans G, Bostrom A. Risky business: challenges in vaccine risk communication. Pediatrics 1998; 101:453–458. 192. Covello VT, Sandman PM, Slovic P. Guidelines for communicating information about chemical risks effectively and responsibly. In: Mayo DG, Hollander RD, eds. Acceptable evidence: science and values in risk management. New York, NY: Oxford University Press, 1991:66–90. 193. Witte K. Putting the fear back in fear appeals: the extended parallel process model. Commun Monogr 1992; 59:329–349. 194. Wolfe RM, Sharp LK, Lipsky MS. Content and design attributes of antivaccination web sites. JAMA 2002; 287(24):3245–3248. 195. Breiman RF, Zanca JA. Of floors and ceilings—defining, assuring, and communicating vaccine safety. Am J Public Health 1997; 87:1919–1920. 196. Salmon DA, Haber M, Gangarosa EJ, et al. Health consequences of religious and philosophical exemptions from immunization laws: individual and societal risk of measles. JAMA 1999; 282:47–53. 197. Salmon DA, Omer SB. Individual freedoms versus collective responsibility: immunization decision-making in the face of occasionally competing values. Emerg Themes Epidemiol 2006; 3:13. 198. Salmon DA, Omer SB, Moulton LH, et al. Exemptions to school immunization requirements: the role of school-level requirements, policies, and procedures. Am J Public Health 2005; 95 (3):436–40. Erratum in: Am J Public Health 2005; 95(4):551. 199. Cohn V. Vaccines and risks: the responsibility of the media, scientists, and clinicians. JAMA 1996; 276:1917–1918. 200. Danovaro-Holliday MC, Wood AL, LeBaron CW. Rotavirus vaccine and the news media. JAMA 2002; 287:1455–1462.

15 Manufacturing of Vaccines Guido Dietrich Berna Biotech Ltd., Berne, Switzerland, and GlaxoSmithKline Biologicals, Wavre, Belgium

Julie Milstien Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Suresh Jadhav Serum Institute of India Ltd., Pune, India

Peter C. Stowe Wyeth Biotech, Division of Wyeth Pharmaceuticals, Collegeville, Pennsylvania, U.S.A.

INTRODUCTION This chapter discusses the production of active preventive vaccines. The manufacturing of passive vaccines (i.e., antisera) or therapeutic vaccines is not covered. The currently licensed active vaccines protect against diseases caused by viral and bacterial infections. There are three basic types: (i) live attenuated vaccines, (ii) killed vaccines (containing the entire bacteria or virus), and (iii) subunit vaccines (containing only certain antigens of the pathogens, which are either derived directly from the pathogen or expressed through recombinant technology). Table 1 divides the most commonly used vaccines into product classes and summarizes the basic techniques involved in their bulk manufacture, with examples of each. Section II outlines the basic production process and defines in more detail what is involved in each of these steps. Section III covers good manufacturing practice (GMP) needed to assure consistency of manufacture; section IV covers some special circumstances; section V new trends in vaccine manufacturing; and section VI projections for the future.

MANUFACTURING PHASES AND CRITICAL ISSUES FOR EACH Introduction The elements of vaccine manufacturing include l

l l l

l l

the receipt and control of raw materials to be used in the production process; the production of purified bulk antigen; formulation of the final bulk; aseptic filling, labeling, and final packaging depending on the composition of the product; analytical capacity to evaluate the products as required; and appropriate storage and distribution.

The entire process may take from one to two years to manufacture and release a vaccine (1), although production can be fast-tracked, for example, for biodefense products or the annual formulation of influenza vaccines.

Raw Materials All raw materials must come from reliable sources and be shown to meet specifications. Most manufacturers use supplier qualification coupled with some quarantine release tests for this purpose. The primary raw materials for biological products, such as seeds, cells, and biological fluids are subject to intense documentation and testing measures, to ensure both consistent characteristics and freedom from adventitious or potentially harmful agents.

Bulk Manufacture Production should occur in dedicated laboratory suites with biologically inactive (prior to infection or after inactivation) areas segregated from production areas in which active materials are being processed. Physical barriers and pressurized air locks are used to prevent cross-contamination of the product; in addition, air and effluents must not contain viruses or bacteria that could contaminate the environment. Seed Preparation Master and working seed bank systems are generally used to initiate manufacture. The bacterial or viral seed is obtained with a well-documented history and is stored in an appropriate manner. Bacterial Fermentation Although fermentation generally refers to the process of growing bacteria in large-volume-closed systems, fermentation

This is a revision and update of the chapter in the previous edition of this volume: Milstien J, Stephenne J, Gordon L. A primer on large-scale manufacture of modern vaccines. Chapter 89. In: Levine MM et al., eds, New Generation Vaccines. New York: Marcel Dekker Inc., 2004:1081–1091.

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Table 1 Process of Bulk Manufacture for Selected Vaccine Types Vaccine type

Process

Examples

Attenuated microbial cells

Growth and purification of microbial cells adapted or engineered to delete pathogenicity, retaining immunogenicity. Steps include fermentation (in flasks or bioreactors), purification, and lyophilization

BCG, oral typhoid, oral cholera

Killed microbial cells

Growth, inactivation, and purification of microbial cells. Steps include fermentation, inactivation, and purification

Whole cell pertussis, anthrax, cholera

Live attenuated viruses

Growth of cells (from cell banks of continuous cells or primary cells from animal/egg tissue), infection with attenuated virus, isolation and purification of virus. Cells are propagated in bioreactors, roller bottles, hollow fiber, cell cubes, flasks, or microcarrier culture with various types of feeding. The medium is generally entirely synthetic but may contain blood proteins to enhance growth. Cells are infected with the vaccine virus; removal of cell or cell debris by centrifugation or ultrafiltration methodology; purification of virus if required, concentration

Yellow fever, measles-mumpsrubella, oral poliovirus vaccine, varicella, rotavirus

Killed viruses

Growth of cells (from cell banks of continuous cells or primary cells from animal/egg tissue), infection with virus, isolation, inactivation and purification of virus. Cells are propagated in bioreactors, roller bottles, hollow fiber, cell cubes, flasks, or microcarrier culture with various types of feeding. The medium is generally entirely synthetic but may contain blood proteins to enhance growth. Cells are infected with the vaccine virus; removal of cell or cell debris by centrifugation or ultrafiltration methodology; purification of virus, if required, concentration; inactivation may occur before or after purification, testing for inactivation

Inactivated poliovirus vaccine, rabies, hepatitis A, Japanese encephalitis B, influenza,

Purified polysaccharides

Growth of bacterial culture, extraction and purification of capsular polysaccharides by centrifugation or filtration; chemical extraction and in some cases, chromatography; chemical characterization of the polysaccharide; concentration and in some cases, drying of bulk

Meningococcal polysaccharide (a/c/w135/y), pneumococcal polysaccharides

Conjugated polysaccharides

Growth of bacterial culture, extraction and purification of capsular polysaccharides, preparation and purification of carrier protein. Chemical modification of polysaccharide; linker if required; chemical processing of carrier, if required; conjugation; separation of conjugated from unconjugated species by chromatography; concentration of bulk conjugate if required

Meningococcal conjugates, Haemophilus influenzae type b conjugate, pneumococcal 7- and 10-valent conjugates

Purified protein, excreted or cell associated

Growth of bacteria, yeast or cell culture where cells are expressing a recombinant protein, cell lysis (for cell associated proteins), isolation and purification of the protein by ultrafiltration methodology; protein purification by chromatography, concentration, buffer exchange, sterile filtration

Hepatitis B (recombinant or plasma derived), human papillomavirus (recombinant), bacterial toxoids, acellular pertussis, split influenza

Live microbial vector

Live attenuated bacteria containing added gene of interest; fermentation in defined media; recovery of whole microbial cells by centrifugation/washing or ultrafiltration methodology

Attenuated salmonella, shigella, Vibrio cholerae, and BCG as vectors for various antigens

Live viral vectors

Growth of cells, infection with genetically engineered replicating nonpathogenic viruses containing added gene of interest, isolation and purification of virus. Cell culturing in bioreactors, roller bottles, hollow fiber, cell cubes, flasks, or microcarrier culture with various types of feeding; virus infection; cell controls; removal of cell or cell debris by centrifugation or ultrafiltration methodology; purification of virus if required

Modified vaccinia ankara, canarypox, adeno, AAV vectors for an assortment of antigens

DNA vaccine

Extraction and purification of plasmid DNA from bacterial cells containing desired gene in the plasmid. Fermentation in defined media; recovery of whole microbial cells by centrifugation/washing or ultrafiltration methodology; cell lysis and removal of cell debris (filtration, centrifugation or expanded bed chromatography); removal of host impurities, RNA, genomic DNA, proteins, and endotoxins (salting out, PEG precipitation); concentration (ultrafiltration methodology, PEG precipitation); purification of plasmid DNA by IEC and/or SEC; concentration and buffer exchange; sterile filtration of final bulk

HIV candidates in DNA plasmids, other candidates

Abbreviation: BCG, bacillus Calmette-Gue´rin.

Chapter 15: Manufacturing of Vaccines

technology has also been used for eukaryotic cell culture. To initiate culture, aliquots of the seed are first grown in a small volume, and the culture is incubated at increasingly larger volumes up to the volume of the final production-scale fermenter. Modern fermenters control and document environmental variables such as temperature, oxygen pressure, and pH enabling standardized production methods to be used. Cell Culture A master cell bank (MCB) consists of frozen vials of a cell line (generally kept in liquid nitrogen), manufactured at as low a passage number as reasonably possible. Documentation includes their origin and their passage histories, the number of passages since origination, storage, and cell culture conditions. Continuous cell lines are usually cloned from a single cell before an MCB is generated to assure purity of the cell line. The stock cultures in the MCB are used to generate the working cell bank (WCB), which is then extensively tested. Cell expansion can progress from flasks to 1 to 10 L spinner cultures and, for large-scale production, to 50 to 10,000 L bioreactor systems. Harvest The mode and method of harvest depends on the product being grown. For lytic viruses, the virus can be intracellular or lysed into the cell culture medium. For the latter, the cell culture supernatant is collected at different time points during production, sometimes over a few days. Perfusion systems where the spent culture supernatant is continuously harvested are popular. Purification After harvesting, the antigens are concentrated and purified using standard techniques such as centrifugation or ultrafiltration. Sometimes, no purification step is performed. For largescale production systems, the virus can be concentrated using ultrafiltration, column chromatography, or gradient ultracentrifugation methods, followed by a sterile filtration step, which can be before or after inactivation. Inactivation Inactivation of purified antigens may be done chemically using reagents such as formaldehyde or peroxide, possibly in combination with heat. Inactivation parameters must be standardized and validated to ensure complete and consistent inactivation.

Formulation and Filling The acceptable limits of variation for factors that impact consistency, such as the amount of antigen, stabilizers, adjuvants, pH, and volume, are ultimately related to the safety and efficacy demonstrated in clinical studies. The three steps of formulation, filling, and freeze-drying are the most critical steps of the entire production process regarding sterility. The reason is that most vaccines, especially those based on whole organisms or those incorporating adjuvants such as alum, cannot be terminally sterilized, in contrast to the majority of drugs and large-volume parenteral solutions. In addition, appropriate mixing of the vaccine must be ensured, in particular in the case of combination vaccines containing antigens against several different pathogens. The length of time of the filling process (e.g., over the course of several hours) must be validated. The definition of a lot, ‘‘doses that are at the same risk of contamination,’’ depends on the capacity of the filling process.

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Packaging of a vaccine product may vary depending on the market. All packaging operations must also be under strict control, and the processes must be documented.

Testing It is critical that biological materials used to generate products for human use are properly qualified. Qualification can include testing for identity, presence of any adventitious viruses, or microbial contamination. Safety assessments, such as lack of reversion for live attenuated vaccines or inactivation for killed vaccines, are also necessary and routine to demonstrate that the product itself remains safe. Furthermore, the amount of antigen is followed over the process to ensure efficacy. Testing is done at multiple steps in the production process. All tests must be standardized and validated. Although the manufacturer has the responsibility for testing and quality assurance procedures on a lot-by-lot basis, all vaccine lots are subject to release by the relevant regulatory authority. In some cases, the release applies to bulk products, in others to the final container vaccine. In either case, time for lot release must be programmed into the manufacturer’s planning process.

Storage and Shipping Storage and shipping of vaccines are also subject to documentation and validation since heating or freezing can damage their integrity. The temperatures, storage, and shipping conditions must be such that the product is stable, and these conditions must be reproducible. Generally, vaccines are stored and shipped using a ‘‘cold chain’’ to maintain temperatures close to or below freezing, depending on the product. Manufacturers use temperature-monitoring devices to ensure that these temperatures are maintained. Real time data on the shipping process must be obtained to ensure product integrity.

GOOD MANUFACTURING PRACTICES Basic Components of Good Manufacturing Practice Compliance with the principles and guidelines of GMP is a statutory requirement applying to all pharmaceutical products, including vaccines. The GMP regulations govern those parts of quality assurance that ensure medicinal products are consistently produced and controlled to the quality standards appropriate to their intended use. Simply stated, production operations must follow clearly defined procedures. In addition, GMP requires evidence of prevention of cross-contamination in production; the performance of validation studies supporting the facilities, systems, processes, and equipment; control of starting materials; control of packaging materials; and handling of finished products. All aspects of these measures and methods of control are specifically defined by local regulations. The term ‘‘cGMP’’ (current GMP) is part of a bigger system of quality assurance in the pharmaceutical industry. Quality assurance as a whole includes all subjects related to ensuring consistency of practice in all phases of a particular endeavor. Issues related to GMP need to be handled right from inception of the manufacturing process. It is the primary responsibility of the regulatory affairs/quality assurance department in a manufacturing facility to ensure that all manufacturing operations performed for production of human vaccines are in compliance with current GMP

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guidelines and follow all regulatory and safety procedures. Some specific responsibilities of this department could include review of standard operating procedures (sops) and batch/ production records; investigation into any procedural or operational error and deviations from sops and/or batch/production records; system inspections/audits; regular cGMP training to personnel; and validation of facilities, equipment, and testing procedures. Validation is the demonstration that a piece of equipment, process, or test performs according to specification so that the data or the products generated are credible.

Applications Facilities Facility design should provide for unidirectional flow of materials, product, and personnel. Production suites should be on separate heat, vacuum, and air conditioning (HVAC) systems that provide classified, preferably HEPA (high-efficiency particulate air filter)-filtered air. Airflows need to be balanced within the production areas to maintain the classification. There should be designated ‘‘clean’’ corridors for entry into production areas. Personnel are required to gown in pressurized entry air locks before accessing the production suites. Exit from production areas should also be through dedicated air locks, where degowning can occur prior to exiting to the return corridor. If possible, separate personnel and material air locks should provide access to the production areas. All systems must be qualified or validated to ensure that they function to specifications, and sops must be present to define the use of all facilities. Containment of microorganisms must also be achieved to avoid their release into the environment. Biological wastes require special equipment for decontamination prior to release. The word ‘‘facility’’ includes, besides manufacturing and testing, the areas for generating various utilities such as water, gases, and steam. It is essential to understand the requirements of facility design in the light of the type of product to be manufactured, the quality of product to be handled, and the type of equipment and the conditions required to achieve desired output.

Environmental Conditions for Production of Vaccines. The manufacture of sterile pharmaceutical products including vaccines needs specified environmental categories to minimize the risk of contamination by microorganisms or particulate matter. There are four general category classifications. Grade a: The local zone of high-risk operations (which involves filling, stoppering, exposure of open ampoules or vials, or making aseptic connections). Usually provided using a laminar airflow work station with a homogeneous air velocity of 0.45 m/sec þ 20% at the working position. Grade b: This is usually a background environment for grade a. Grades c and d: For operations that are of less critical stages in the manufacture, but still require higher environmental conditions than those existing normally in the laboratory. The airborne particulate classification for these grades is given in Table 2. Not only are pharmaceutical products vulnerable to the presence of particulate matter they are also vulnerable to bacterial contamination. Air forced through HEPA filters keeps the bacterial contamination generated by the operators away from the product in a class a area. The background

Table 2 Airborne Particulate Classifications At resta

In operation

Grade

Maximum permitted number of particles/m3 equal to or above

a bb cb db

0.5 mm 3500 3500 350,000 3,500,000

5.0 mm 0 0 2000 20,000

0.5 mm 3500 350,000 3,500,000 Not definedc

5.0 mm 0 2000 20,000 Not definedc

a The guidance given for the maximum permitted number of particles in the ‘‘at rest’’ condition corresponds approximately to the U.S. Federal standard 206 e and the ISO classifications as follows: grades a and b correspond with class 100, m 3.5, ISO 5; grade c with class 10,000, m 5.5, ISO 7, and grade d with class 100,000, m 6.5, ISO 8. b To reach the b, c, and d air grades, the number of air changes should be related to the size of the room and the equipment and personnel present in the room. The air system should be provided with appropriate filters such as HEPA for grades a, b, and c. c The requirement and limit for this area will depend on the nature of the operations carried out.

Table 3 Recommended Limits of Microbial Contamination

Grade a b c d

Settle plates (diameter Air sample 90 mm), cfu/m3 cfu/4 hra <1 10 100 200

<1 5 50 100

Contact plates (diameter Glove print 55 mm), 5 fingers cfu/plate cfu/glove <1 5 25 50

<1 5 – –

These are average values. a Individual settle plates may be exposed for less than four hours. Abbreviation: cfu, colony-forming units.

Table 4 Examples of Activities in Classified Areas Grade a c d Grade a c d

Examples of operations for terminally sterilized products Filling of products, when unusually at risk Preparation of solutions, when unusually at risk. Filling of products Preparation of solutions and components for subsequent filling. Examples of operations for aseptic preparations Aseptic preparation and filling Preparation of solutions to be filtered Handling of components after washing

environmental condition, that is, class b for class a, would remove the generated particles and bacteria from the clean environment back to the air-handling unit via the return air duct. Manufacturers should monitor microbiological units during operations (Table 3). To achieve the prescribed environmental and cleanliness standards, the prescribed manufacturing process needs to be classified into activities on the basis of the cleanliness of operations (Table 4).

Chapter 15: Manufacturing of Vaccines

Additional standards are as follows: l

l

l

l

Gowning. There are guidelines that describe the requirements for respective areas and the type of gowning recommended. Grade d. Hair and, where relevant, beard should be covered. A protective suit with either shoes or overshoes is recommended. Grade c. All conditions mentioned in grade d plus a single or two-piece trouser suit closing at neck and wrist. The fabric chosen to prepare these gowns is expected not to shed fibers or particulate matter (lint-free cloth). Grades a and b. All requirements mentioned in d and c plus headgear, a face mask, along with foot covers, gloves, and covering of eyes with spectacles. The garments used in classes a and b areas must be sterilized, and it is advisable not to make repeated use of them.

Pharmaceutical water. Water is a major component solvent used in all manufacturing processes; as a part of the final product its characteristics are critical. In the pharmaceutical industry, water is defined according to its quality as in Figure 1. Normally, water received from metropolitan supplies may contain residual chlorine, although it is potable. Thus it is chemically impure. It is first deionized and subsequently ultrafiltered, or passed through the reverse osmosis system, to yield highly purified water. This water is also used as the feed water for generating distilled water and, finally, water for injection (WFI), which is kept continuously flowing through the distribution loop at a temperature around 808C. Since this water needs to comply with very high standards of purity and microbiological content, an elaborate water-testing system is needed. Equipment such as total organic carbon analyzers, which can be fitted on line in the circulation loop, allow monitoring of the WFI quality. Pure steam, which is generated using WFI or highly purified water, must be used for sterilizing all product contact parts. After condensation, it should comply with all the tests performed on WFI. System-related elements. Deviation control system Any change in a sop, which is an unplanned activity, must be reported to quality assurance immediately as a deviation, where its impact on product quality and safety is evaluated. Change control system A planned deviation is categorized by an expert committee as critical or noncritical (type 1 or type 2). This committee also reviews the impact of such a change on safety and efficacy of product. Corrective action preventive action Issues related to the points observed in internal audits or external audits in the manufacturing and testing areas need to be addressed in a timely manner so that they do not recur. The corrective action preventive action (CAPA) system follows up these actions. Other system-related issues include audits (internal/ external), training, and sop writing.

Figure 1 Schematic indication of water quality.

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Equipment Equipment used in production should be installed, operated, and maintained as per cGMP guidelines. Where appropriate, Installation/Operational/Performance Qualification (IQ/OQ/ PQ) protocols, calibration, and validation studies need to be designed and executed. All equipment should be maintained on current calibration and preventive maintenance schedules performed as recommended by their manufacturers. Documentation All operations in GMP manufacturing, whether dealing with facility, equipment, material usage, manufacturing, or product release must be performed as outlined in the SOPS. This ensures that the manufacture is performed in strict accordance with established manufacturing, regulatory, and safety procedures. Any deviation from sops has to be reported, and its effect on the clinical product documented. Each sop is uniquely identified, usually stored in both electronic and paper formats, and maintained by the document control department. Production or batch records are used to document all manufacturing operations that are performed in the production of human vaccines. These records are established in accordance with appropriate sops, and are distributed and controlled by the document control department. Any deviations from manufacturing operations outlined in the sops are documented in the appropriate batch records. Batch records are especially important when it becomes necessary to revisit the manufacturing operations, if some inconsistency or deficiency is discovered in the clinical product. Validation protocols must be maintained on file as part of the documented record of a production process. Process and quality control validation are becoming more important to confirm the robustness of the methods employed.

SPECIAL CIRCUMSTANCES IN VACCINE MANUFACTURING Although manufacturing processes and practices have advanced with the development and application of new technology, certain critical vaccines are still manufactured by traditional methods because of lack of suitable substitute technology, or lack of incentive for developing improved technology. An example is the smallpox vaccine, which was manufactured by methods originally developed in the late 18th century, until the increased concern of bioterrorism in the aftermath of the anthrax bioterror events that followed the September 11, 2001, airliner hijackings in the United States led to a new demand for this vaccine. After a 30-year hiatus in efforts to develop an improved process, government solicitation of bids from manufacturers willing to produce a new version of the vaccine resulted in the awarding of the supply contract to a company committed to providing vaccine produced by a new in vitro tissue culture manufacturing process, which substantially improved purity and speed to market.

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Influenza vaccine, which has been in continued global demand for well over a half century, has continued to be manufactured by methods fundamentally unchanged over that period of time. The antigen is cultivated on a commercial scale in vivo, in the allantoic fluid of embryonated chicken eggs, and chicken protein remains as a trace impurity in the vaccines. Although the traditional method of manufacturing the vaccine has proven practical and effective, the complexity and variability of certain elements of the process leave manufacturers susceptible to unexpected disruptions to the supply of suitable eggs for this process, and to virus strains that do not propagate well in the allantoic fluid. Although demand for this vaccine has increased, the usual disincentives to process improvements are further complicated by the seasonal nature of influenza, and the variable risk associated with the continued ‘‘antigenic drift’’ and ‘‘antigenic shift’’ of this pathogen. Thus, the vaccine is often nonefficacious for new strains of the virus during the following season. The prediction of influenza pandemic may however provide an incentive for process change such as the introduction of new in vitro cell-based processes. At the other end of the spectrum, there are diseases for which the entire array of vaccine manufacturing options, both traditional and modern, have been ineffective for either largescale or small-scale quantities of vaccine. An example of this is Neisseria meningitidis, serogroup B, which causes devastating, often fatal clinical infections. Because the natural antigenic elements of the bacteria (the polysaccharide capsule) emulate human nervous tissue, and may elicit an autoimmune response when administered as a traditionally prepared vaccine, this vaccine provides a unique challenge for those involved in efforts toward developing an effective vaccine for this disease.

NEW TRENDS IN VACCINE MANUFACTURING In recent years, there are a number of novel tools and trends that are pursued in pharmaceutical manufacturing, and also taken up by vaccine manufacturers. These include novel ways of managing the manufacturing operations, such as operational excellence initiatives and tools, as well as new types of disposable equipment.

Operational Excellence The most important operational excellence trends include lean manufacturing, Total Quality Management (TQM), quality risk management, Six Sigma, and Process Analytical Technology (PAT). Lean manufacturing originated from the automotive industry (2), but has recently been taken up by pharmaceutical companies. The key elements of lean manufacturing are avoiding waste and concentrating on adding value. Throughout a process, all steps are analyzed whether they add value to the product (from the standpoint of the customer), and every step, action, and practice that does not create value is eliminated. The remaining steps occur in a tight and integrated sequence so that the product will flow smoothly toward the customer. TQM is based on the goal that all processes of the value chain are designed to achieve optimal quality. It requires total management commitment and a philosophy of excellence. Consequently, TQM is highly focused on management and improvement of all processes within the value chain. Quality risk management originated from industries such as aerospace engineering, nuclear power plant design, and the

automotive industry. Its integration in the operations of pharmaceutical companies (including vaccine manufacturers) was initiated mainly by the recently released International Conference on Harmonisation (ICH) Q9 guideline on risk management standards (3). Regulatory authorities such as the European Medicines Agency (EMEA) (4) and Food and Drug Administration (FDA) (5,6) are taking up the initiative, but have not yet made the utilization of risk management techniques mandatory. The four basic steps of risk management are (i) risk identification, (ii) risk evaluation, (iii) risk control and mitigation, and (iv) risk monitoring (verification of risk reduction). Nevertheless, it must be clearly recognized that there is generally some level of residual risk with all products, and it will never be possible to eliminate or mitigate all risks! Six Sigma stands for the standard deviation manufacturers want to achieve within their processes and products. The main characteristic of Six Sigma is the strong adherence to statistical analysis of all processes. By achieving a smaller standard deviation, the processes will be more predictable, will generate less waste and rework, and will yield products that perform better, and thereby enhance customer satisfaction. When analyzing processes with six sigma, there are five distinct steps to follow, the so-called DMAIC (define, measure, analyze, improve, and control) cycle: Define: the process is defined, Measure: the process inputs and outputs are measured, Analyze: all data measured are being analyzed, Improve: as a result of the analysis, the process can be improved, and Control: it is verified that the improvements indeed yield a better process. PAT was launched by FDA and is defined as a system for designing, analyzing, and controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality (7). FDA emphasizes tools for scientific, risk-managed pharmaceutical development, manufacture, and quality assurance. These tools are categorized as (i) multivariate data acquisition and analysis tools, (ii) modern process analyzers or process analytical chemistry tools, (iii) process endpoint monitoring and control tools, and (iv) continuous improvement and knowledge management tools. Use of these tools is expected to result in continuous quality improvements. When processes are so stable that the predefined product quality levels are always achieved, PAT may even allow real time release of products. The growing importance and use of these and other operational excellence tools and philosophies reflects that more and more vaccine companies consider product manufacturing as an important asset, for which optimal strategies have to be defined and pursued.

Disposables in Vaccine Manufacturing Disposable materials have been used for many years in blood collection and blood processing, so there is a huge body of experience with such materials. In vaccine manufacturing operations, however, the more limited use of disposables has been expanded only recently. Disposables have numerous advantages. They come pre-packed and presterilized. Their single use eliminates the need for cleaning and cleaning validation, and reduces the risk of carryover from one lot to the next during a manufacturing campaign, or between different products in multipurpose facilities. Changeover times can be dramatically reduced. Use of disposables can also make the validation of production processes for new vaccines easier and

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faster, allowing shorter time to market. Finally, disposable systems reduce the need for large-scale cleaning and sterilization equipment, and the costly plant utilities needed to operate that equipment. A facility designed on disposables use was recently found to be less expensive, faster to build and qualify, and easier to maintain (8). The use of disposable materials does, however, require that appropriate quality control and quality assurance procedures be followed. It is the responsibility of the vaccine manufacturers to ensure that the disposables used are qualified for their processes, and that their manufacturing processes are validated when disposable materials are implemented (9). Solid-waste disposal is another issue to be addressed. In upstream processes of vaccine manufacturing, disposable materials can be used in the preparation and intermediate storage of media and buffers (10), but more importantly, disposables are currently used as bioreactors for the growth of viruses and even bacteria. Recently, more sophisticated bioreactors were developed, including hollow fiber bioreactors and bag-based systems (11). Systems that are fully integrated with disposable sensors and sampling systems are being developed (11). In downstream processing, disposable filters and chromatography membranes greatly facilitate cleaning validation, showing in particular the advantages of disposable materials. Disposable filters may also replace more complex technologies in manufacturing processes, for example, size exclusion chromatography may be replaced with ultrafiltration in the purification of conjugate vaccines (12). Applicable to all steps of the vaccine manufacturing process (including aseptic filling) are new opportunities to make sterile connections. Disposable technologies are also the basis for tube welders and sealers, which allow for sterile connections and disconnections, respectively (13).

THE FUTURE The Increasing Value of the Vaccine Market The value of the vaccine market has been steadily increasing, from an estimated $2 billion in 1982 (14) to $25.2 billion currently (15), and is projected to be $50.7 billion by 2013 (15), according to a recent analysis by Kalorama Information. Part of this increase reflects an expansion of the traditional pediatric target population, so that more children are receiving a wider array of vaccines. However, most of the increase results from the introduction of pediatric ‘‘blockbuster’’ vaccines such as the pneumococcal conjugate vaccine, which has an earnings record of nearly $2 billion per year, comparable to the most profitable chemical medicines (16) and from increased uptake of influenza and hepatitis vaccines in adults. A major increase is expected from a new role for cancer prevention vaccines, expanding from hepatitis b vaccines to the human papillomavirus vaccines against cervical cancer, and to new vaccines under development against a variety of other cancers (15).

Rising Vaccine Production Capacity in Developing Country Manufacturers The increased potential revenue in the vaccine market, especially related to newer technologies, has resulted in a shift in the vaccine manufacturers providing to the global public market, that is, the so-called UNICEF market—that proportion of the vaccine market devoted to providing vaccines for public sector immunization programs. This market has historically been of low value compared to the proprietary market. Figure 2

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Figure 2 2004 vaccine sales by market segment. Source: UNICEF Supply Division Annual Report (2000–2004) and IMS Health Incorporated (BCG 2005).

shows that division in 2004 taken from a recent study of the vaccine industry (17) conducted by the Boston Consulting Group (BCG) for partners of the GAVI alliance (GAVI). The increased revenue potential in other vaccine markets has resulted in an increasing proportion of the multinational companies (MNCs) leaving the global public sector market. Their place has been taken by emerging large public and private sector companies in Asia and Latin America. This can be seen by the proportion of vaccines prequalified for WHO for UN agency procurement (18): in 1986, 50% of prequalified vaccine products came from emerging suppliers; in 1996, 67%; and in 2006, 71%. The increasing percentage of manufacturing for the global public market, coupled with the increase in quality and access to technologies for these manufacturers, will have a large impact on the pediatric vaccine market itself, as well as on the position of these emerging vaccine manufacturers on that market. The study cited above (17) included 17 manufacturers from seven countries, Brazil, China, Cuba, India, Indonesia, Republic of Korea, and Mexico, and concluded that among these, there were about six from countries in Latin America and Asia, which were likely to have a strong role in providing innovative vaccines in the future.

Rising Vaccine Technological Capacity in Developing Country Manufacturers The BCG study (17) also looked at the ability of these manufacturers to access technologies increasingly used in vaccines of special interest today, and of potential use for tomorrow. In the 17 emerging manufacturers studied, there were 58 products in R&D of interest to GAVI, compared to 22 GAVI priority products among the six MNCs studied. A subsequent analysis of the Indian health biotech sector (19) concluded that a set of local vaccine manufacturers (including those in the BCG study) ‘‘leverage revenues from the sale of (familiar) vaccines to develop more innovative vaccines,’’ and they have also made

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the large-scale processes used in production of these products more efficient, thus making them more cost-competitive.

Increasing Partnerships among MNCs and Developing Country Manufacturers Partnership agreements may be the fastest route to develop a product, and to get around issues of access to technologies and know-how; on the other hand, they may limit markets and the flexibility of the participants. For the MNC partners, they may provide a potentially lower-cost production site. Most MNC partnership agreements start with allowing filling and finishing of their bulk vaccine by the emerging supplier partner. Of the seven manufacturers studied in two countries in Asia and Latin America, five had technology transfer agreements with MNCs, accounting for a significant proportion of their pipeline products (19). Some of these manufacturers have developed MNC partnerships for other biological products besides vaccines. It was seen that those manufacturers deemed stronger by other analyses were involved in expanding their technological capabilities to a wider variety of biological products, in some cases, seeking to enter regulated markets through registration of such biopharmaceutical products. Frewe et al. (20) cited the establishment and maintenance of collaborations and partnerships with both public and private organizations by Indian biotech firms as a way to establish a global presence and to assure transfer of technologies and knowledge in both directions.

2. 3. 4.

5.

6. 7.

8. 9.

10.

11. 12. 13.

Projections As the vaccine market evolves, what will be the future roles of MNCs relative to emerging suppliers?a We have seen that both the MNCs and, to a lesser extent, the emerging suppliers, are accessing new technologies for use in vaccine manufacture. In another chapter in this volume (13), it will become apparent that emerging suppliers can no longer be differentiated from MNCs on the basis of quality. This also implies that the price differential between the two groups will be less important, and more dependent on economies of scale in areas where labor costs are not the significant drivers. A major difference may be product selection. Emerging suppliers have often been driven by national and regional needs in product development, and thus may become sites for regionally specific products.

ACKNOWLEDGMENT We would like to thank Marie Lovoll for critical reading of the manuscript.

14.

15.

16. 17.

18.

19.

20.

REFERENCES

New Generation Vaccines. 3rd ed. New York: Marcel Dekker Inc., 2004:75–87. Womack JP, Jones DT, Roos D. The machine that changed the world. New York: Rawson, 1990. ICH Harmonised Tripartite Guideline. Available at: http://www. ich.org/lob/media/media1957.pdf. Accessed October 2007. European Commission. Working party on control of medicines and inspections. Available at: http://ec.europa.eu/enterprise/ pharmaceuticals/eudralex/vol-4/pdfs-en/v4an15.pdf. Accessed October 2007. Available at: http://www.fda.gov/Drugs/DevelopmentApprovalProcess/Manufacturing/QuestionsandAnswersonCurrentGoodManufacturingPracticescGMPforDrugs/UCM071836. Accessed September 2009. Available at: http://www.fda.gov/cder/gmp/gmp2004/ risk_based.pdf Accessed September 2009. Available at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070305.pdf. Accessed September 2009. Wassard K, Monge M. Rapid implementation of a smallpox vaccine facility. Bioprocess Int 2007; 5(suppl 4):32–37. Scott C. Disposables qualification and process validation. Managing risk to address leachables, extractables, and more. Bioprocess Int 2007; (suppl 4):24–27. De Waele K, Vandermarliere M, von Broekhoven A, et al. A novel single-use mixing system for buffer preparation. Bioprocess Int 2007; (suppl 4):56–61. Scott C. Single-use bioreactors. Bioprocess Int Suppl Series 2007; (suppl 4):44–51. Pora H. Improving speed and quality in vaccine production. Pharm Technol Europe 2007; 19(3):33–34. Kinney SD, Phillips CW, Lin KJ. Thermoplastic tubing welders and sealers. Bioprocess Int 2007; (suppl 4):52–55. Milstien J, Candries B. Economics of vaccine development and implementation: changes over the past 20 years. The Jordan Report 20th Anniversary. Accelerated Development of Vaccines, U.S. Department of Health and Human Services, National Institute of Allergy and Infectious Diseases, 2002:73–79. Available at: http://www.prlog.org/10232681-the-worldwidemarket-for-vaccines-to-reach-US507-billion-in-2013-forecastsnew-report.html. Accessed September 2009. Wyeth 2008 Financial Report (2008). Company Data by Reportable Segment (p. 43). GAVI. Global vaccine supply: the changing role of suppliers. Available at: http://www.gavialliance.org/resources/BCG_24_June.pdf. Accessed: October 2007. Milstien JB, Dellepiane N, Belgharbi L, Chocarro L, Wood D. Assuring Vaccine Quality by Strengthening Regulatory Agencies: The Work of the World Health Organization. In: Levine MM, Dougan G, Good MF, et al., eds. New Generation Vaccines. 4th ed. New York: Informa Healthcare USA, Inc. 2009:121–130. Milstien JB, Gaule P, Kaddar M. Access to vaccine technologies in developing countries: Brazil and India. Vaccine 2007; 25(44): 7610–7619. Frewe SE, Rezaie R, Sammut SM, et al. India’s health biotech sector at a crossroads. Nat Biotechnol 2007; 21(4):403–417.

1. Greco M. Development and supply of vaccines: an industry perspective. In: Levine MM, Kaper JB, Rappuoli R, et al., eds.

a Note that this study has not looked at the role of the biotech industry in general, nor its specific impact on biological products, including therapeutic vaccines. It is entirely possible that the biologics industry of the future may include entirely different classifications than those we deal with in this chapter.

16 Polio Eradication: Ongoing Innovation to End an Ancient Scourge R. Bruce Aylward Department of Vaccines and Biologicals, World Health Organization, Geneva, Switzerland

Stephen L. Cochi Global Immunization Division, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

David L. Heymann London School of Hygiene and Tropical Medicine, London, U.K.

INTRODUCTION By the time the Global Polio Eradication Initiative (GPEI) was launched in 1988, humans had a long and intimate knowledge of this devastating disease, as evidenced by its first-known depiction in an Egyptian funereal stele dating from 3000 B.C. (1). Our knowledge of eradication, however, was much younger, dating only to the early 1900s when U.S. Army General Gorgas embarked on his ill-fated quest to eradicate yellow fever from the jungles of Panama (2). By 1988, two more eradication initiatives had failed, against yaws and malaria (3,4), while the only one that has so far proven successful, against smallpox, did so by the slimmest of margins (5). Despite eradication’s mixed record of success historically, when the World Health Assembly (WHA) decided to pursue polio eradication in 1988, it did so with confidence that it understood the disease, that the available tools could interrupt transmission of poliovirus everywhere, and that society’s dread of paralytic polio was strong enough to ensure sufficient financing and community mobilization to implement the strategies everywhere. For more than 10 years the WHA’s confidence proved well founded (6). Although the GPEI was behind schedule by the late-1990s due to insufficient financial and political backing, most public health officials concurred that interruption of wild poliovirus globally was indeed feasible, and many presumed it would soon be followed, at least in some countries, by cessation of immunization against the disease, as had been the case with smallpox (7). Ironically, it has only been since 2000, the original target date for polio eradication, that the world has appreciated new challenges to interrupting poliovirus transmission worldwide and securing a polio-free world for future generations. Though sobered by these challenges, the GPEI has responded with a range of innovative solutions. By early 2007, the GPEI had new tools, new tactics, and new commitments, leading Dr Margaret Chan, the newly elected Director-General of the World Health Organization (WHO), to present a compelling case for completing polio eradication to the WHA on May 17, 2007 (8). This chapter summarizes the rationale underpinning the polio eradication effort, the history of the GPEI through the

year 2000, the nature and impact of the new challenges and innovations that have emerged since then, and the strategies currently under discussion to eliminate any residual paralytic poliomyelitis due to continued use of the oral poliovirus vaccine (OPV) in the ‘‘post-eradication’’ era.

THE RATIONALE FOR POLIO ERADICATION Although the framework now used for evaluating the ‘‘eradicability’’ of a given disease was developed only in the late-1990s, it provides a useful structure for summarizing the rationale that underpinned the decision to eradicate polio (9,10) (Table 1).

Technical Feasibility By the late 1980s, polio was deemed to be eradicable from a biologic and technical perspective because of l

l

l

an effective intervention and delivery strategy that could interrupt transmission, practical diagnostic tools with sufficient sensitivity and specificity, and absence of a nonhuman reservoir.

Polio eradication was deemed biologically feasible because humans are essential for the life cycle of the virus, which has no other reservoir and does not amplify in the environment (11). Although polio has been described among orangutans, chimpanzees, and gorillas in captivity and chimpanzees in the wild, these species appear to be incidental hosts with populations too small to sustain transmission. Similarly, viable virus cannot be found in sewage or surface water for more than several weeks after circulation ceases among humans. Although two excellent polio vaccines have existed for many decades [inactivated polio vaccine (IPV) has been licensed since 1955 and OPV since the early 1960s] and IPV had been used to interrupt poliovirus in three industrialized countries of northern Europe (Finland, the Netherlands, Sweden), OPV had proven a more suitable intervention for

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Table 1 Poliomyelitis and the 1997 Dahlem Eradication Criteria Criteria for targeting a disease for eradication Biological and technical feasibility Etiologic agent Nonhuman reservoir Effective intervention tool Effective delivery strategy Simple/practical diagnostic Sensitive surveillance Field-proven strategies Costs and benefits Cases averted per year Coincident benefits Intangible benefits Estimated annual direct global savings Estimated total external financing Societal and political considerations Political commitment (endemic/industrial countries) Societal support (endemic/industrial countries) Disease burden in politically unstable areas (% cases from war-torn countries) Spearheading partners Technical consensus Donor base (number of donors of US$ 1 million or more by 2006)

Poliomyelitis Virus No Oral polio vaccine Mass immunization via national immunization days Stool culture Facility-based active surveillance Western hemisphere 650,000 Improved immunization and surveillance capacity Culture of prevention and social equity US$ 1.5 billion US$ 8 billion Strong/variable Variable/strong <5% (estimated) World Health Organization, Rotary, US Centers for Disease Control and Prevention, United Nations Children’s Fund, Bill and Melinda Gates Foundation World Health Assembly

the developing country setting (12). By the mid-1980s, there was strong evidence from Cuba and other countries of Latin America, especially Brazil, that OPV delivery through mass campaigns could stop transmission in tropical climates, probably because of the induction of superior secretory intestinal immunity compared with IPV, the more rapid increase in population immunity, and the protection of close contacts of vaccinees (13,14). Furthermore, the low cost and simple practical oral administration route of OPV made it much more suited to a mass delivery strategy. Establishing a sensitive and specific system for polio diagnosis was complicated as the majority of wild poliovirus infections (>99%) are subclinical (15) and even when there is paralysis, polio cannot be definitively confirmed clinically (16,17). Serologic testing is inadequate because it cannot distinguish antibodies due to wild polioviruses from those produced by vaccine. Consequently, the GPEI has relied on poliovirus culture from stool specimens for diagnosis (18), which although resource intensive has high specificity, sensitivity, and predictive value. Most importantly, by 1988 there was ‘‘proof of concept’’ that poliovirus could be interrupted over a large geographical area as evidenced by the progress toward regional elimination of the disease from the Western Hemisphere [WHO Region of the Americas or Pan-American Health Organization (PAHO)] (19,20).

Benefit: Cost Ratio A crucial element of the argument for eradicating polio is that the marginal costs of moving from control to eradication can soon be recouped through savings because of foregone treatment and control costs as well as increased productivity (21). Although economic analyses of disease eradication are problematic because of the lack of consensus on how to value benefits that accrue in perpetuity (22), several studies have estimated the costs and benefits of polio eradication. In 1994, it was estimated that annual global savings would exceed US$1.5

billion per year once polio had been eradicated and all control measures stopped (23). A more recent analysis found that even if universal IPV was used following polio eradication, the cost per DALY saved would still be less than US$50 (24). In 2007, as the marginal costs of polio eradication escalated because of the low number of cases, a new study assessed the rationale for further investments (25). This study found that irrespective of long-term policy decisions (i.e., whether to use OPV or IPV in a post-eradication era), low-income countries alone stand to save billions of dollars and to prevent up to 4 million paralyzed children over the next 20 years if polio eradication is completed, compared with shifting to a polio control program. The indirect benefits of polio eradication were central to the launching of the initiative in the Americas (26) as well as the WHA resolution that endorsed global eradication (27). By end2006, the GPEI investment had already paid major dividends beyond the prevention of 5 million polio cases. The GPEI had helped: avert 1.25 million deaths through vitamin A supplementation and through end-2007 2.6 million measles deaths were averted (28); boost routine immunization coverage and introduce new vaccines in Global Alliance for Vaccines and Immunization (GAVI)-eligible countries; manage international health emergencies such as Severe Acute Respiratory Syndrome (SARS) and Avian Influenza and novel H1N1 influenza (29); and respond to humanitarian crises such as the South Asia Tsunami in 2004 and the Pakistan earthquake in 2005.

Societal and Political Support for Eradication Although the successful conclusion of the smallpox eradication campaign in 1977 created some momentum for new eradication efforts, this enthusiasm was countered by concerns that targeted objectives compromise efforts to develop primary health care systems (30). Within the scientific community some doubts as to the technical feasibility of polio eradication (31) persisted despite the progress in the Americas. Notwithstanding these

Chapter 16: Polio Eradication

concerns, substantial political and societal will existed to support embarking on global eradication, galvanized by Rotary International, the volunteer service organization whose 1.2 million members in 140 countries would donate over US$800 million of the US $8 billion in external financing expended on the initiative as of end-2008 (32). The GPEI has in general enjoyed tremendous international goodwill and political commitment; by end-2000 every country in the world, including those with the scarcest of resources, had introduced the necessary strategies and over 60 countries, foundations, nongovernmental organizations and companies had provided external financing. Political leaders have repeatedly demonstrated their role by participating in highly visible events such as the launching of national immunization days (NIDs).

A BRIEF HISTORY OF POLIO ERADICATION THROUGH THE YEAR 2000 In 1985, the success of polio control efforts in a number of Latin American countries spurred the PAHO Regional Director to establish a regional goal of polio elimination by 1990. By 1988, PAHO had refined its strategy into the four-pronged approach that would form the basis for the global effort (20): (i) routine immunization to optimize immunity against polio by ensuring as high a proportion of children as possible received three (subsequently increased to four) OPV doses as early as possible in

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infancy; (ii) annual NIDs to interrupt the major chains of indigenous poliovirus transmission with two rounds of supplementary OPV immunization, four to six weeks apart during the low season for enteroviruses, targeting all children younger than five years, regardless of their prior immunization history; (iii) virologic investigation through an accredited laboratory network of all cases of acute flaccid paralysis (AFP) in children younger than 15 years, and ‘‘suspect polio’’ in persons of any age, to increase the sensitivity of surveillance for circulating polioviruses (33) using standard indicators to monitor performance (34,35); and (iv) large-scale house-to-house OPV mop-up campaigns to interrupt any remaining chains of poliovirus in a country or area (36). The event that is considered to have been pivotal in transforming the regional PAHO effort into a global initiative was a March 1988 meeting convened by the Task Force for Child Survival and Development, which issued the Declaration of Talloires (37). Only two months later, the Director-General of WHO, who had participated in the meeting, put the matter to a vote at the WHA, which consequently endorsed a polio eradication resolution. Although surveillance data were incomplete, estimations based on reported cases globally and lameness surveys in selected countries indicated that well over 350,000 children were still being paralyzed by poliovirus each year (36). In addition, at least 125 countries (based on 2005 boundaries) were still suffering endemic polio or, in the case of smaller island nations, sporadic epidemics of the disease (Fig. 1). The

Figure 1 Known and suspected distribution of indigenous wild poliovirus in 1988, the year the Global Polio Eradication Initiative was launched, and at end-2008.

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Figure 2 Network of laboratories for diagnosis of vaccine-preventable diseases that has been built on the Global Polio Laboratory Network, as of June 2007.

eradication goal was subsequently endorsed by the World Summit for Children in 1990, the largest ever gathering of Heads of State (38). Political leaders from low-, middle-, and high-income countries have continued to reconfirm their commitment through resolutions adopted in summits that include those of the G8, African Union (AU), which was preceded by the Organization of African Unity (OAU) (39), Organization of Islamic Conferences (OIC), and the South Asian Association for Regional Cooperation (SAARC) (40). Following the WHA resolution, rapid progress continued in the Americas, with the last case of indigenous polio occurring in August 1991 in Peru (26). By the mid-1990s, large-scale activities were ongoing in WHO’s Western Pacific (WPR), Eastern Mediterranean (EMR), and European (EUR) regions. In the six endemic countries of WPR, including China, polio was rapidly interrupted, with the last case occurring in Cambodia in March 1997 (41). In EUR and EMR, activities accelerated with Operation MECACAR in which NIDs were synchronized across 18 countries of the MEditerranean, CAucasus, Central Asian Republics and Russia, immunizing 56 million children, three years in a row, beginning in April and May 1995 (42). The last indigenous case of polio in EUR occurred in southeast Turkey in November 1998 (43). By the year 2000, indigenous poliovirus had also been interrupted in 9 of the 10 countries of Southeast Asia, WHO’s most densely populated Region. In India, the sole exception, indigenous polio was restricted to just 2 of the 30 states. The cessation of polio in Indonesia (1995) and Bangladesh (2000) reinforced the effectiveness of the strategies in highly populated areas. By 2001, indigenous poliovirus in EMR was limited to parts of Pakistan, Afghanistan, Egypt, the Sudan, and Somalia. In the WHO Region of Africa, with 46 sub-Saharan countries, largescale activities began with the 1996 ‘‘Polio-free Africa’’ declaration at the OAU, led by Mr. Nelson Mandela. Progress was surprisingly rapid, with indigenous polio interrupted everywhere except Nigeria, Niger, Ethiopia, and Angola by 2001.

The progress between 1988 and 2000 was the result of multiple factors that facilitated strategy implementation (27). There was a strong ‘‘core’’ partnership of four agencies with a long-term commitment of time, expertise, and resources: WHO, Rotary International, the U.S. Centers for Disease Control and Prevention (CDC) and the United Nations Children’s Fund (UNICEF). This partnership mobilized the high-level political commitment that facilitated fundraising and strategy implementation. An enormous investment was made in surveillance, underpinned by a global network of 145 accredited laboratories (44,45) (Fig. 2). Finally, there was a massive deployment of technical assistance (reaching nearly 3500 people in 75 countries at the peak of activities) and investment of external financing (totaling US$8 billion by end-2008) (32,46). Although wild poliovirus transmission had not been interrupted globally by 2000, all countries had introduced the necessary strategies by then, and substantial progress had been made. Most notably, one of the three poliovirus serotypes (type II) had been eradicated globally, with the last virologically confirmed case of paralytic polio due to indigenous type II virus occurring in India in late 1999 (47). Consequently, support for the GPEI was high as it entered the new millennium. Between 2000 and 2003, however, a range of new problems emerged, which challenged the very precepts upon which the GPEI was founded. These challenges resulted in a period of unprecedented innovation, however, so that by 2009 the feasibility of the GPEI again appeared sound: only four countries still had indigenous poliovirus transmission though 11 of the reinfected countries had yet to interrupt polio again.

NEW TOOLS AND TACTICS TO ADDRESS NEW CHALLENGES This section outlines, in order of programmatic importance, the new challenges and problems that emerged for the GPEI since

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Figure 3 Comparison of polio immunization coverage among children aged 6 to 59 months (based on an analysis of non-polio acute flaccid paralysis cases) in areas with continued transmission of indigenous poliovirus at end 2002.

2000, as well as the solutions that have been developed and applied in response.

Areas of Highly Efficient Poliovirus Transmission By mid-2003 it was evident that despite very high OPV coverage, polio transmission was being sustained in areas of Egypt and India where population numbers and density were particularly high and sanitation was suboptimal, such as in greater Cairo, Mumbai, and, especially, a cluster of districts in the western part of the Indian state of Uttar Pradesh (Fig. 3) (48–50). Some questioned whether the eradication strategy itself might be flawed when it came to interrupting wild poliovirus in such settings. In response, the options for enhancing the impact of supplementary immunization activities (SIAs) were reevaluated, focusing on replacing (or supplementing) the trivalent OPV formulation used in SIAs with either IPV or a monovalent OPV (mOPV) targeting the specific poliovirus serotype that was circulating in a particular area. In October 2004, the GPEI’s oversight body recommended WHO pursue the rapid development and introduction of mOPV type 1 (mOPV1), given the substantial gains in immunity it conferred for type 1 poliovirus when compared with tOPV as well as its operational advantages over IPV (51). By mid-2005 two mOPV1s were already licensed and in use in Egypt and India as the result of an extraordinary collaboration between WHO and UNICEF and the vaccine manufacturers Sanofi-Pasteur and Panacea Biotec, with financing from the Bill and Melinda Gates Foundation and under the oversight of national vaccine regulatory agencies. By end-2005 transmission of indigenous wild polioviruses was interrupted in greater Cairo, Upper Egypt, and Mumbai (in Egypt the last virus was detected in January 2005). Although India experienced a large polio outbreak in 2006 due to the accumulation of susceptibles over the previous four years, a case-control study confirmed that in western Uttar Pradesh the per-dose efficacy

of the mOPV1 against type 1 poliovirus was at least twofold that of trivalent OPV (52). Although polio cases fell markedly by mid-2007, the Government of India and the GPEI were also investigating the value of further enhancing the potency of mOPV1 and/or administering one to two doses of IPV with the mOPV1 to further enhance young child immunity in west Uttar Pradesh. On December 25, 2005, mOPV3 was introduced for the first time in the GPEI when 10 million doses were used to respond to persistent circulation of a lineage of type 3 poliovirus in western Uttar Pradesh, India.

Areas of Suboptimal Strategy Implementation In contrast to Egypt and India, continued polio transmission in the other four countries that still had indigenous virus by mid2003 was the result of low OPV coverage in key geographical areas (53). In Pakistan and Afghanistan, transmission was being sustained in remote and sparsely populated areas where substantial pockets of under-immunized children remained because of remote geographic location, active conflict, and even local culture and tradition, which limited the ability of immunization teams to enter the houses to search for and vaccinate any missing children. Substantial cross-border population movements between the two countries allowed the viruses to continuously find susceptible children. In Nigeria and Niger, the problem was much greater as less than 50% of children in the remaining, much larger, infected areas were receiving four or more doses of OPV at that time (Fig. 3). Consequently, in early 2003 new tactics were employed to rapidly raise coverage by increasing the number of polio campaigns, deploying additional technical assistance to improve NIDs microplanning and systematically engaging political, religious, and community leaders. Coverage rose rapidly in three of the countries so that indigenous polio was interrupted throughout Niger and all but the border areas of

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Figure 4 Impact of immunization campaigns with monovalent oral poliovirus vaccine type 1 (mOPV1) on cases of paralytic poliomyelitis due to type 1 poliovirus, Nigeria, January 2006–April 2007.

Afghanistan and the city of Karachi in Pakistan by 2006. In northern Nigeria, however, the increasing profile of the GPEI led some local commentators in mid-2003 to accuse the NIDs of being a plot to sterilize young girls (54). As the debate escalated in the local media, leaders in a number of states decided to suspend all OPV until the concerns could be addressed. Tragically, the OPV suspension dragged out for a full 12 months, resulting in a nationwide polio epidemic in Nigeria and extensive international spread to 20 previously polio-free countries (see below). Even after political leaders backed the safety of OPV in Nigeria, new tactics were required to rebuild community confidence in immunization: Local and international religious and traditional leaders were engaged to promote the NIDs, community dialogues were held before and after each round, and the campaigns themselves were expanded into ‘‘Immunization Plus Days’’ in mid-2006 to better address community demands for a wider range of health interventions. In Afghanistan and Pakistan, the Ministers of Health of both countries announced in November 2006 the synchronization and coordination of all polio campaigns to ensure the remaining crossborder reservoirs of virus were interrupted. Beginning in 2006, mOPV1 replaced trivalent OPV in many of the campaigns in all three countries, with a particularly impressive impact in Nigeria (Fig. 4).

Reinfection of Previously Polio-Free Areas Until 2003, the inevitable international spread of polio from endemic areas was programmatically manageable as it was usually limited to importations into just two to four polio-free areas each year. However, the 12-month OPV suspension in parts of northern Nigeria resulted in unprecedented international spread of polio over the subsequent 36 months (55); by mid2007, 20 countries had been reinfected at least 70 times by viruses that were genetically linked to those originating in northern Nigeria (Fig. 5). During the same period, another seven countries were reinfected by viruses originating in northern India.

A multipronged approach was taken to limit this international spread of polio: mop-up activities were conducted in reinfected countries; ‘‘preventive’’ NIDs were restarted in countries at high risk of reinfection; multicountry campaigns were coordinated by the AU across 25 countries of west and central Africa; enhanced surveillance was instituted (see below); an international travel alert was issued, recommending full immunization of travelers to and from areas of uncontrolled polio (56); and the newly developed mOPV1 was approved for outbreak control (57) (Fig. 6). In May 2006, the WHA endorsed strong new international guidelines for responding to circulating poliovirus in poliofree areas, markedly increasing the size, speed, and duration of outbreak activities with the appropriate mOPV (58). In May 2007, the WHA went further to recommend that countries update national immunization policies to limit the international spread of polio as Saudi Arabia had done in late 2006, requiring, among other measures, proof of OPV vaccination from all travelers from endemic countries (59,60). By mid-2007, the new outbreaks resulting from this international spread of polio had largely been stopped; however, thousands of children had been paralyzed and over US$450 million was expended in emergency activities.

Gaps in Poliovirus Surveillance While investigating polio cases in the above-mentioned outbreaks, genetic sequencing of viruses detected in the Sudan and Chad suggested that two lineages of wild poliovirus type 3 and one lineage of type 1 had been circulating undetected for three years in the Horn of Africa and central Africa (61). Investigations demonstrated that this was due to a failure to fully implement the AFP surveillance strategy, rather than a failure of the strategy itself—in south Sudan and large parts of Chad, AFP surveillance had never met international standards. The GPEI subsequently undertook a large-scale, systematic effort to achieve and sustain polio surveillance standards at

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Figure 5 International spread of wild polioviruses, January 2003–June 2007.

Figure 6 Countries using monovalent oral poliovirus vaccine type 1 (mOPV1) for the interruption of indigenous or imported polioviruses between April 2005 and June 2007.

the subnational, as well as national, levels in all endemic, reinfected, or high-risk countries. For programmatic purposes, the AFP sensitivity performance standard was increased from a target of one non-polio AFP case per 100,000 children younger than 15 years to two (57). By end-2006, surveillance sensitivity had improved in all areas of GPEI programmatic importance, and genetic sequencing of polioviruses had not uncovered any major new gaps (62). The speed and effectiveness of AFP surveillance was further enhanced in mid-2006 by the introduction of new laboratory processes across the Polio Laboratory

Network, reducing by 50% the time required to confirm a poliovirus infection (63).

Circulating Vaccine-Derived Polioviruses Perhaps most striking of the new challenges that the GPEI encountered was the evidence from Hispaniola in 2000, showing that Sabin strain polioviruses could regain neurovirulence and the capacity to circulate and cause outbreaks (64). Between 2000 and 2009, circulating vaccine-derived polioviruses

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Figure 7 Distribution of seven polio outbreaks caused by circulating vaccine-derived polioviruses between January 2000 and August 2009.

(cVDPVs) caused at least ten additional polio outbreaks in the Philippines (2001) (65), Madagascar (2002, 2005) (66), China (2004), Indonesia (2005), Nigeria (2006–2009), Cambodia (2005-2006), Myanmar (2006–2007), DR Congo (2008), and Ethiopia (2008– 2009) (Fig. 7). To better understand the frequency and extent of cVDPVs, historical poliovirus isolates were screened using an ELISA assay, which typically gave a non-Sabin-like result for cVDPVs. All such viruses were then subject to sequencing of the full VP1 region of the polio genome to determine its nature. Potential markers of VDPV circulation, such as the presence of recombination with a non-polio enterovirus, were exploited to help differentiate cVPDVs from other possible sources such as iVDPVs (a VDPV excreted from an individual with a primary immunodeficiency syndrome) (67). Operational guidelines were established for investigating and, if appropriate, responding to VDPVs. By 2002, standardized methods for screening all isolated polioviruses for VDPVs were introduced into all of the Polio Network laboratories that were conducting intratypic differentiation. Despite the genetic similarities between cVDPVs and wild polioviruses, cVDPVs have thus far proven easier to interrupt, usually requiring two to three subnational trivalent OPV rounds targeting hundreds of thousands of children. By contrast, interruption of wild poliovirus outbreaks during the same period has required on average six to seven rounds of nationwide immunization (55). The interruption of a cVDPV in an area that is very highly conducive to poliovirus circulation, such as western Uttar Pradesh in India, could yet prove substantially more difficult.

SECURING A POLIO-FREE WORLD The global cessation of smallpox immunization soon after the eradication of its causative pathogen in 1977 contributed greatly to the widespread expectation that polio immunization would stop soon after eradication of wild polioviruses (68). However, there are important differences in the vaccines used to eradicate each of these diseases, as well as the political circumstances of the periods in which each initiative was

conducted. The smallpox vaccine was made from the vaccinia virus rather than the smallpox virus itself and caused a very high rate of severe side effects (i.e., up to 1 severe adverse event per 25,000 doses administered) (69). Furthermore, the disease was eliminated at a time when concerns about its potential deliberate use to cause harm were much less than they are today. Until the year 2000, discussions on long-term polio immunization policy were primarily driven by the humanitarian and economic benefits of OPV cessation. In particular, it was widely felt that once wild poliovirus had been interrupted globally, the public health benefits of OPV would no longer outweigh the estimated 250 to 500 cases of vaccine-associated paralytic poliomyelitis (VAPP) that would continue to occur each year on the basis of current vaccine utilization patterns (70). As progress toward global polio eradication advanced in the late 1990s and the risk of wild poliovirus importations declined, industrialized countries began switching from OPV to IPV for routine childhood immunization to avoid VAPP (71), despite a higher cost-benefit ratio for IPV (72). More recently, cVDPVs have taken on a greater significance than VAPP for long-term polio immunization policy. Such episodes demonstrate that after wild poliovirus eradication the use of OPV would continually generate cVDPVs, the spread of which could eventually reverse the eradication achievement. Recognizing this, expert committees have since the late 1990s recommended eventual, simultaneous cessation of all routine OPV immunization, as soon as possible after confirmation of wild poliovirus eradication (51,57,73,74).

The Major Risks Associated with OPV Cessation While there are clear benefits to eventually stopping OPV, these must be weighed against the associated risks of human infection, and subsequent transmission, following ingestion of a Sabin strain, VDPV, or wild poliovirus in the post-eradication era. Sabin strain polioviruses are ubiquitous due to their use in millions of OPV doses each year in over 150 countries as well as

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Figure 8 Risks associated with eventual cessation of routine childhood immunization with oral polio vaccine (OPV), by income bracket (in upper-middle income countries, risks and policies often reflect high-income countries; in low-middle income countries, risks and policies often reflect low-income countries).

for OPV seed viruses, reference standards, diagnostic test controls, basic research, and teaching. While the vast majority of OPV recipients will lead to time-limited (i.e., 3–4 weeks) shedding of the virus, Sabin viruses can give rise to cVDPVs or, very rarely, an iVDPV (see below). Of particular importance is the danger posed by the emergence of a cVDPV(s) immediately after countries stop using OPV. Mathematical modeling suggests that even with simultaneous OPV cessation there is a 60% to 95% chance of at least one cVDPV outbreak in the world during the 12 months immediately after cessation, although that risk declines to 1% to 6% at 36 months and much lower thereafter (75). VDPVs have drifted genetically by at least 1% from the parent, usually Sabin, strain and can arise from prolonged replication of a vaccine strain in an individual or circulation in a population (cVDPV). Prolonged VDPV excretion (>6 months) is rare and almost always occurs in individuals with certain primary B cell–related immunodeficiency syndromes (iVPDVs). None of the 42 iVDPVs confirmed by August 2009 led to secondary cases of paralytic polio, although there has been at least one case of asymptomatic infection of contacts (76). Twenty-one of the individuals with iVDPVs have spontaneously stopped excreting, died, or were lost to follow-up; five iVDPVs excreted chronically (WHO working definition >60 months), two of whom are known to have continued excretion as recently as 2005. Acquired immunodeficiency syndromes involving T cells, such as with human immunodeficiency virus (HIV) infection, have not been associated with prolonged poliovirus excretion (77). Although extremely rare, ‘‘chronic’’ iVDPVs take on a special importance in the context of OPV cessation, as they might subsequently reinfect an increasingly susceptible human population (78,79), and there is not yet an effective antiviral therapy or other proven strategy to eliminate infection. Wild polioviruses are currently used as seed viruses for IPV production, in vaccine quality control and assurance testing, in diagnostic test controls, and for research. Although wild polioviruses are no longer nearly as ubiquitous as Sabin viruses, and no evidence exists of their long-term carriage, the consequences of an inadvertent or intentional release in the post-OPV era pose a far greater threat. A recent consequence assessment suggests that whereas transmission of a Sabin strain virus may or may not be self-limiting in an unvaccinated

population in the post-OPV era, a wild poliovirus would almost always result in a large-scale outbreak with a real risk of eventually reestablishing transmission globally (79). Figure 8 summarizes, on the basis of current knowledge, the expected evolution of these risks in low- and high-income countries over a probable three to five year ‘‘OPV cessation’’ phase following confirmation of wild poliovirus interruption and appropriate biocontainment of all poliovirus stocks globally.

Risk Reduction and Management A comprehensive approach must be taken to manage the risks associated with OPV cessation (80). The core principles are to (i) stop simultaneously the routine use of OPV worldwide, then recall and destroy remaining stocks; (ii) reduce the number of poliovirus procedures to those which are essential in the postOPV era; (iii) replace wild polioviruses with Sabin strain viruses for procedure(s) that must continue in the post-OPV era [including IPV production, (81)]; (iv) minimize the number of sites handling or storing any polioviruses or potentially poliovirus-infectious materials, and limiting these sites to geographical areas where the consequences of an inadvertent release could be minimized (79); (v) ensure residual poliovirus sites fully implement appropriate biocontainment and biosafety procedures (82); (vi) maintain surveillance to identify and monitor VDPVs and to detect the release of any poliovirus; and (vii) establish a stockpile of mOPVs, and possibly IPV, with internationally agreed criteria for their use in mounting typespecific outbreak responses in the post-OPV era. OPV cessation will require establishing international concurrence, through a body such as the WHA, to apply these core principles in all areas of all countries in the world, with additional activities in areas that pose particular risks. Areas with large, high-density populations and low routine immunization coverage may be at high risk for generating cVDPVs and need an OPV pulse or even IPV to boost immunity beforehand. Countries that store or handle polioviruses in the post-OPV era will constitute an international biohazard and may require extraordinary measures to prevent, or minimize the consequences of, inadvertent virus release (83). Among the unresolved issues regarding OPV cessation is the degree to which IPV should be used to minimize the

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Figure 9 Districts in which wild poliovirus transmission was detected between March 30th, 2009 and September 29th, 2009.

associated risks. Although universal childhood immunization with IPV has been proposed to address the risks associated with OPV cessation, IPV would only partially reduce the already small risk of cVDPV emergence in most countries (75) and would not substantially mitigate the consequences of a reintroduction in countries with low coverage, such as much of sub-Saharan Africa (84). Consequently, policy makers must balance their national willingness to pay to maintain polio immunity against the financial, programmatic, and opportunity costs of introducing IPV, the true extent of which may not be immediately apparent, particularly for resource-poor areas (81). In financial terms alone, UNICEF currently procures IPV at five times the estimated ‘‘breakeven’’ price for replacing OPV (85). Even if a marginal reduction in the unit price of IPV for low-income countries materializes, the opportunity costs associated with the use of scarce health resources for that vaccine (e.g., rather than to combat HIV, malaria, tuberculosis, measles, pneumococcal, and rotavirus infections) would strongly influence decision making. Some low income countries have already decided that the advantages of stopping all polio immunization currently outweigh the short-term risk of cVDPVs and longer-term risks of poliovirus reintroduction (86). In contrast, some middle-income countries are introducing one or more doses of IPV into their routine immunization schedules to eliminate VAPP and as a potential transition strategy between OPV cessation and verification of the absence of cVDPVs.

POLIO ERADICATION: THE UNFINISHED AGENDA Since the GPEI’s launch in 1988, knowledge as to the nature of circulating polioviruses and the challenges to their interruption has increased tremendously, particularly during the period 2000 to 2006. By mid-2009, however, indigenous wild polioviruses had been interrupted from all but four countries in the world

though 16 reinfected countries still had circulation of an imported virus (Fig. 9). Recent progress reflects the rapid development and large-scale application of new tools (e.g., mOPVs) to improve the impact of the traditional strategies, coupled with new tactics and new political commitments to ensure that every child is vaccinated. The proof of concept in mid-2009 of a bivalent OPV (serotypes 1 and 3), with per dose seroconversion rates that are almost equivalent to those for the respective type-specific mOPV, promises to further enhance the impact of SIAs and potentially accelerate eradication in the remaining infected areas. Although the prospects of eventual success for the GPEI are high, research must continue to further evaluate and enhance the strategies, particularly in northern India where additional refinements may be required to boost immunity to the levels needed to stop transmission. Similarly, while the long-term risks of polio are much better understood than when the GPEI started, continued research is needed to optimize the strategies for their management. For example, the frequency, magnitude, and consequences of iVDPVs must be determined in low- and middle-income countries. The effectiveness and cost/benefit of IPV costreduction strategies (i.e., 2-dose schedules and fractional dosing) must be evaluated for low-income countries. New, rapid diagnostics should be pursued as well as antiviral compounds to clear iVDPVs. mOPV campaigns in low-coverage areas are being studied to explore whether mOPV use in a post-OPV era might give rise to new cVDPVs. Finally, insights into whether cVDPVs can be stopped with an IPV response would help to inform outbreak response strategies for a postOPV era. At mid-2009, the primary challenges to a world without polio are ensuring that local leaders in each remaining polioinfected district guarantee the full vaccination of their children, and that national and international leaders ensure the financing needed to implement fully the eradication strategies (Fig. 10).

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Figure 10 International and major national financial contributions and pledges to the Global Polio Eradication Initiative, 1988–2009* (US$7.94 billion), and funding gap for planned activities in 2010.

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Chapter 16: Polio Eradication 70. Vaccines & Biologicals Department. Report of the interim meeting of the Technical Consultative Group (TCG) on the Global Eradication of Poliomyelitis, Geneva, 13–14 November 2002. Geneva: World Health Organization, 2004. 71. Centers for Disease Control and Prevention. Poliomyelitis prevention in the United States. Updated recommendations of the advisory committee on immunization practices. MMWR Morb Mortal Wkly Rep 2000; 49(RR-5):1–22. 72. Miller MA, Sutter RW, Strebel PM, et al. Cost-effectiveness of incorporating inactivated poliovirus vaccine into the routine childhood immunization schedule. J Am Med Assoc 1996; 276(12): 967–971. 73. Technical Consultative Group to the World Health Organization on the Global Eradication of Poliomyelitis. ‘Endgame’ issues for the global polio eradication initiative. Clin Infect Dis 2001; 34:72–77. 74. Vaccines and Biologicals. Report of an informal consultation on the identification and management of Vaccine-derived Polioviruses (VDPVs). Geneva: World Health Organization, 2004. 75. Duintjer Tebbens RJ, Pallansch MA, Kew OM, et al. Risks of paralytic disease due to wild or vaccine-derived poliovirus after eradication. Risk Anal 2006; 26:1471–1505. 76. Centers for Disease Control and Prevention. Poliovirus infections in four unvaccinated children—Minnesota, August–October 2005. Morb Mortal Wkly Rep 2005; 54(Dispatch):1–3. 77. Hennessey KA, Lago H, Diomande E, et al. Poliovirus vaccine shedding among persons with HIV in Abidjan, Cote d’Ivoire. J Infect Dis 2005; 192:2124–2128.

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78. Kew OM, Wright PF, Agol VI, et al. Circulating vaccine-derived polioviruses: current state of knowledge. Bull World Health Organ 2004; 82:16–23. 79. Fine PEM, Ritchie S. Perspective: determinants of the severity of poliovirus outbreaks in the post eradication era. Risk Anal 2006; 26:1533–1540. 80. Polio Eradication Initiative. Cessation of routine Oral Polio Vaccine (OPV) use after Global Polio Eradication. Framework for National Policy Makers in OPV-using Countries. Geneva: World Health Organization, 2005 . 81. Heymann DL, Sutter RW, Aylward RB. A global call for new polio vaccines. Nature 2005; 434:699–700. 82. Dowdle W, van der Avoort H, de Gourville E, et al. Containment of polioviruses after eradication and OPV cessation: characterizing risks to improve management. Risk Anal 2006; 26:1449–1469. 83. World Health Organization. Inactivated poliovirus vaccine following oral poliovirus vaccine cessation: supplement to the WHO position paper. Wkly Epidemiol Rec 2006; 81:137–144. 84. World Health Organization. Introduction of inactivated poliovirus vaccine into oral poliovirus vaccine-using countries: WHO position paper. Wkly Epidemiol Rec 2003; 28:241–250. 85. Sangrujee N, Ca´ceres VM, Cochi SL. Cost analysis of post-polio certification immunization policies. Bull World Health Organ 2004; 82(1):9–15. 86. Griffiths UK, Botham L, Schoub BD. The cost-effectiveness of alternative polio immunization policies in South Africa. Vaccine 2006; 24:5670–5678.

17 Recent Advances in Immunology That Impact Vaccine Development Marcelo B. Sztein Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Rafi Ahmed Emory Vaccine Center, Emory University School of Medicine, Emory University, Atlanta, Georgia, U.S.A.

Shane Crotty La Jolla Institute for Allergy & Immunology, La Jolla, California, U.S.A.

INTRODUCTION The first decade of this millennium witnessed an explosion in information concerning the mechanisms underlying the host’s immune response to invading microorganisms, as well as during pathological conditions such as autoimmune diseases and cancer. This was largely due to technological advances that include (i) the ability to track specific T cells by using fluorochrome-labeled peptide-major histocompatibility complex (MHC) tetramers and B cells, by using fluorochrome-labeled antigens (Ag); (ii) sophisticated multichromatic (up to 17 colors) and flow cytometric sorting techniques that allow the identification and functional characterization of subpopulations of cells on the basis of the concomitant expression of multiple surface and intracellular molecules (e.g., homing molecules, cytokines); (iii) the availability of an ever-growing number of cytokines, chemokines, and other immunoregulatory molecules produced by recombinant techniques; (iv) gene expression profiling using DNA microarrays that allows simultaneous determination of the expression of thousands of individual genes in response to infection; (v) ‘‘systems biology’’ approaches to predict the magnitude of the immune responses; (vi) the advent of proteomics (i.e., the large-scale analysis of proteins and their interactions); and (vii) engineering of a multitude of single and multiple gene knockout and transgenic animals (and more recently ‘‘conditional knockout’’ and ‘‘knockin’’ mutants). Moreover, the availability of the DNA sequences of entire genomes from an ever-increasing number of eukaryotic and prokaryotic organisms has the potential to identify large numbers of novel target Ag for vaccine development. Despite the knowledge gained in recent years, there is no consensus on a general set of critical immunological principles that should guide the development of new vaccines. On the basis of the information available to date, it is likely that the desired immunological responses that must be elicited by effective vaccines will have to be tailored to each individual pathogen. This is due to the complexity of the immune response, the diversity of pathogenic microorganisms, the various routes of entry into the host, and the ability of some invading pathogens to subvert the generation of protective immune responses.

Consequently, it is difficult to select ‘‘a priori’’ among the many factors that influence the development of a successful vaccine, such as the choice of the appropriate ‘‘protective’’ Ag(s), route of administration, dose, immunization schedule, adjuvants, and formulation. It is also difficult to predict accurately how the age and the immunological status of the recipient will affect responses to the vaccine. This review intends to highlight, rather than discuss in depth, recent advances in our understanding of the mechanisms underlying the generation of immune responses and immunological memory. Emphasis will be placed on those mechanisms that have directly advanced (or have the potential to advance) the development of new vaccines or can improve existing vaccines. This chapter will succinctly summarize novel concepts on the integrated nature of innate and adaptive immunity and will focus on recent findings in the areas of Ag processing and presentation (including costimulatory molecules), the role of cytokines and chemokines in linking adaptive and innate immunity, the molecular determinants underlying lymphoid cell trafficking, and the generation of memory B and T lymphocytes. Because of space limitations, the reader will be referred throughout this chapter to excellent recent reviews for additional information.

AN INTEGRATED VIEW OF THE IMMUNE RESPONSE The host’s immune defense mechanisms against infectious agents can be divided into two main components: innate (i.e., native immunity) and adaptive (i.e., acquired immunity). Both of these complementary and highly interrelated components of the immune system are essential to protect the host from disease-causing viruses, bacteria, fungi, and parasites.

Innate Immunity This component of the immune response plays a critical role immediately after a pathogen enters the host, providing a first line of defense against invading microorganisms. Innate

Chapter 17: Recent Advances in Immunology That Impact Vaccine Development

immunity is primarily responsible for the elimination, or at least the control, of the invading microbe during the four to seven days required for the establishment of an early adaptive immune response (1–5). Moreover, it is now widely recognized that innate immunity and the resulting inflammatory process play a key role in initiating the adaptive immune response and determining its nature. A distinctive feature of innate immunity is that the response does not increase with successive exposures to the microbes. However, of note, a recent study has described a ‘‘memory’’ response by natural killer (NK) cells following infection with mouse cytomegalovirus (6). Many cells are involved in innate immunity, including phagocytes (e.g., neutrophils, macrophages), dendritic cells (DC), NK cells, and eosinophils. A key characteristic of the cells that form part of the innate immune system is their ability to recognize a wide range of microorganisms through surface receptors (pattern recognition receptors [PRRs]) that recognize invariant molecules present in a wide range of microbes (pathogen-associated molecular patterns [PAMPs]) but not in the host (1). This important area of research has received a remarkable degree of attention over the past few years (1–5,7–10). Examples of PAMPs, which are present in both pathogenic and nonpathogenic microorganisms, include

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bacterial cell wall peptidoglycans of gram-positive bacteria and lipopolysaccharide (LPS) of gram-negative bacteria. PRRs can be expressed on the cell membrane, in intracellular compartments or secreted (2–5,7–10). Examples of PRRs expressed on the cell surface include the macrophage scavenger receptor (MSR) that recognizes polyanionic ligands (e.g., dsRNA, LPS), lectin-like binding receptors on NK cells, and macrophage mannose receptors (MMR) that recognize carbohydrate structures present in bacteria and fungal pathogens. Intracellular PRRs include the protein kinase R (PKR) and the 20 -50 -oligoadenylate synthase, which binds dsRNA (present in viruses), as well as the Nucleotide-binding and oligomerization domain containing proteins (NOD), which appear to respond to LPS. Secreted PRRs (e.g., C-reactive protein, mannan-binding lectin, etc.) function by binding to microbes, leading to their elimination by complement-mediated mechanisms or phagocytosis. Toll-like receptors (TLR) are an important family of PRRs that play a pivotal role in innate immune recognition (2–5,7–10). TLR are characterized by extracellular domains that contain leucine-rich repeats and cytoplasmic portions, responsible for intracellular signaling, similar to the intracellular domain of the type 1 interleukin (IL)-1 receptor. Thirteen murine TLR and 10 human TLR, which recognize a variety of different PAMPs, have been

Table 1 Summary of the Properties of Toll-Like Receptors in Humans TLR

Cell location

Major ligands/agonists

TLR1 (þ TLR2)

Surface

Bacterial triacyl lipopeptides

Gram-positive bacteria

TLR2

Surface

Peptidoglycan (PGN) Lipoarabinomannan (mycobacteria) HSP60 Bacterial lipoproteins/lipopeptides GPI anchor (Trypanosoma cruzi) Glycolipids Phenol-soluble modulin (Staphylococcus) Zymosan (Saccharomyces cerevisiae) LPS from Porphyromonas gingivalis and Leptospira interrogans HA (Hemagglutinin, measles)

Gram-positive and gramnegative bacteria Mycobacteria Mycoplasma Protozoa Fungi Virus

TLR3

Intracellular (endosomal)

dsRNA Poly I:C

Viruses

TLR4

Surface

Enterobacterial LPS Lipoteichoic acid (LTA) HSP60, HSP70 Respiratory syncytial virus F protein Teichuronic acid (Micrococcus luteous) Bacterial fimbriae (Escherichia coli) Taxol Extra domain A (EDA) of fibronectin Fibrinogen Hyaluronic acid Heparan sulfate

Gram-negative and grampositive bacteria Chlamydia Viruses

TLR5

Surface

Flagellin

Gram-positive and gramnegative bacteria

TLR6 (þ TLR2)

Surface

Bacterial diacyl lipopeptides

Gram-positive bacteria Mycoplasma

TLR7

Intracellular (endosomal, lysosomal?) Intracellular (endosomal, lysosomal?)

ssRNA (e.g., HIV) Antiviral drugs (imidazoquinoline) ssRNA Antiviral drugs (imidazoquinoline)

Viral and nonviral ssRNA

TLR9

Intracellular (endosomal, lysosomal?)

Unmethylated CpG DNA motifs Viral DNA?

Bacteria Viruses

TLR10

Surface

Unknown

TLR8

Note: "?" stands for unknown. Abbreviations: LPS, lipopolysaccharide; HSP, heat-shock protein. Source: From Refs. 2–5 and 7–10.

Microbes

Viral and nonviral ssRNA

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described to date (Table 1) (2–5,7–10). Of note, TLR sometimes require other molecules to participate in the recognition of PAMPs. For example, TLR4 has to be coordinately associated with MD-2 and CD14, as well as CD11b/CD18 heterodimers, to enable optimal LPS signaling, leading to nuclear translocation of nuclear factor kB (NF-kB) (7), and TLR2 has to dimerize with other TLR, such as TLR1 or TLR6, to detect ligands and induce signaling (4,5). Recognition of ‘‘microbial nonself’’ through PRRs’ recognition of PAMPs triggers signaling pathways that result in activated phagocytes that are better equipped to engulf and destroy the offending pathogen, as well as to secrete many molecules responsible for the initiation of an inflammatory process. Molecules secreted following TLR-PAMP interactions include inflammatory cytokines (e.g., interferon [IFN]-a, IFN-b, IL-1a, IL-1b, IL-6, IL-12, and tumor necrosis factor [TNF]-a), chemokines (e.g., CXCL8 [IL-8], CCL20 [macrophage inflammatory protein (MIP)-3a], CXCL10 [IP-10], and CCL2 [MCP-1]), and other moieties that directly or indirectly participate in the destruction of the microbe. As discussed in more detail below, these molecules profoundly affect the behavior of many cell types, modulate the type of adaptive immune response induced, and recruit macrophages, neutrophils, DC, lymphocytes, and other cells to the inflammatory site. Of note, TLRPAMP interactions on the surface of DC, a key Ag-presenting cell (APC) (e.g., through TLR7, TLR9; (5)), play a critical role in the induction of adaptive immunity by upregulating the expression of costimulatory molecules required for the activation of naive T cells specific for antigenic peptides expressed on the same DC in conjunction with MHC molecules (1). It is also important to emphasize that cytokines and other molecules secreted by cells of the adaptive immune system (e.g., IFN-g secreted by activated T lymphocytes) have, in turn, marked effects on the innate immune response through activation of macrophages and other cells that ultimately participate directly in the destruction of the microbe. The importance of TLR in innate and adaptive immunity is further underscored by recent evidence from genetic association studies in humans suggesting that TLR polymorphisms may be associated with disease susceptibility or protection (11). Inflammasomes Host cell interactions with a variety of microbial stimulants, including TLR ligands, result in the initiation of inflammatory responses through the formation of ‘‘inflammasomes.’’ Inflammasomes are large multiprotein complexes whose assembly leads to the activation of caspase-1, which in turn promotes the processing and secretion of proinflammatory cytokines, in particular three IL-1 family members: IL-1b, IL-18, and IL-33 (12–14). Upon cleavage of their proforms by caspase-1, these cytokines become active and are secreted. These cytokines are critical for the regulation of adaptive immunity. For example, IL-1b and IL-18 are potent proinflammatory cytokines. IL-18 induces IFN-g expression and secretion from IL-12-primed naı¨ve T cells to promote the differentiation of T helper (Th)1 cells. In contrast, IL-33, through binding of the IL-1 receptor family protein ST2, promotes Th2-mediated responses (15). Caspase-1 has been shown to play a critical role in inflammation and in the pathogenesis of several autoinflammatory diseases such as the cryopyrin-associated periodic syndromes (CAPS) and rheumatoid arthritis. These insights have provided critical information for understanding and treating these autoinflammatory disorders. In fact, these diseases have been effectively treated by the administration of IL-1 receptor

antagonists, supporting the central role of IL-1 in their pathogenesis (12). There are at least two major inflammasomes, that is, the Apaf-1 and Nacht leucin-rich repeat protein 3 (NALP-3) inflammasomes (12). Proteins encoded by these nucleotidebinding domain and leucine-rich repeat (NLR) containing gene families form the central components of inflammasomes and act as intracellular sensors to detect cytosolic microbial components and ‘‘danger’’ signals (such as ATP and toxins) (12–14). The inflammasome not only plays a pivotal role in innate immune responses toward pathogens but also mediates the activity of aluminum adjuvants. Thus, the inflammasome and associated signaling pathways are attractive targets for new therapeutics and vaccines (13). Antigen-Presenting Cells The ability of cells to function as effective APC depends on their ability to process Ag for class I MHC-restricted cytotoxic T lymphocyte (CTL) responses and/or class II MHC-restricted Th responses (16). Since most of the cells of the body express class I MHC molecules and have the ability to express on the cell surface endogenously produced peptides complexed to class I MHC molecules, they have the potential to function as APC for CD8þ CTL. In fact, cells that are endogenously producing viral, parasitic, or bacterial proteins or tumor Ag that gain access to the cytosol can be recognized and destroyed in a class I MHCrestricted fashion by specific CTL. However, while all class I MHC-expressing cells can be recognized and killed by effector CD8þ cells, non-APC are inefficient in activating naı¨ve CD8þ cells. Only professional APC efficiently initiate the differentiation program that leads naı¨ve T cells to develop into effector and long-lived memory CD8þ T cells (see below for details). In contrast, the main characteristics of APC required for presentation to Th cells in a class II–restricted fashion are the ability to take up soluble Ag from the extracellular compartment and process them to produce appropriate peptides that will then be complexed to class II MHC molecules and expressed on the cell membrane for recognition by Th cells. The cells that most efficiently present Ag to Th lymphocytes, the so-called ‘‘professional APC,’’ include DC, macrophages, and B lymphocytes. Most professional APC express moderate to high levels of class II MHC molecules constitutively, and their expression can be upregulated upon activation by cytokines such as IFN-g. Moreover, professional APC express many costimulatory and adhesion molecules, which are very important during the early stages of T-cell activation. In contrast, ‘‘nonprofessional APC,’’ such as endothelial, epithelial, and mesenchymal cells, typically do not express class II MHC molecules constitutively but can be induced to express them following exposure to T cell–derived cytokines, such as IFN-g. Their role as APC in vivo is still unclear. Dendritic Cells A large body of evidence accumulated over the past decade clearly demonstrates that DC are the most effective APC involved in activation of naive T cells (16–22). Because of their key role in the induction of immunity, a thorough understanding of DC biology is of paramount importance in vaccine development. DC comprise a heterogeneous cell population that originates in the bone marrow from hematopoietic stem cells and then reside, as immature DC, largely in peripheral tissues exposed to the environment, that is, the sites of Ag entry. In the absence of ongoing inflammation and immune responses, DC’s main function is to be vigilant for invading microbes in

Chapter 17: Recent Advances in Immunology That Impact Vaccine Development

both lymphoid and nonlymphoid tissues (e.g., secondary lymphoid tissues, skin, blood, lymph, and mucosal surfaces). Immature DC express receptors for inflammatory chemokines that direct their migration to sites on inflammation. Upon recognition of microorganisms expressing PAMPs, through PRRs, receptors for the Fc portion of Ig and other receptors, exposure to cytokines, chemokines and other inflammatory stimuli, and, under certain circumstances, self-Ag, DC quickly mature into efficient APC and migrate into draining lymph nodes (LN) where they initiate primary T-cell responses (17–22). However, it is important to emphasize that not all DC in LN and spleen originate from DC that have been already exposed to Ag in peripheral tissues. In fact, sizable proportions of DC in the LN and spleen are immature and derived from a blood-borne progenitor (20). The maturation process involves the upregulation of MHC molecules, as well as costimulatory molecules (e.g., CD40, CD54, CD58, CD80, CD86), chemokine receptors (e.g., CCR7), and adhesion molecules, which drive their migration into the lymphatic vessels and the T-cell areas of the draining LN. To reiterate, in addition to presenting Ag, on the basis of the type of cytokines they release and their expression of distinct adhesion/costimulatory molecules, DC play a key role in determining the type of adaptive immunity elicited (e.g., polarized Th1 vs. Th2 responses). In addition, DC have been implicated in the induction of CD4þ Tcell differentiation into alternative cell fates, including regulatory cells or the newly discovered IL-17-producing CD4þ T cells (20). Of note, not all DC that exhibit maturation markers are able to prime naı¨ve T cells. In fact, sometimes they induce tolerance (20). Two main subsets of functionally distinct DC have been described in human peripheral blood, that is, myeloid DC (mDC), also called ‘‘conventional DC,’’ and plasmacytoid DC (pDC), which can be differentiated on the basis of the surface expression of molecules that determine their function (17–24). Both populations of immature DC isolated from human peripheral blood lack lineage differentiation markers, including CD14, CD16, CD19, CD3, and CD56. mDC are lineage negative, CD11cþ, CD123/low, CD4 þ, CD80hi, CD86þ, CD45ROþ, CD45RAlow, CD33þ, CD13þ, CD54þ, CD58þ, CD62L/low, CD36low, CD83þ (small subset, most negative), HLA-DRhi, CD206 (mannose receptor)þ, CCR7, TLR2þ, TLR4þ, TLR7, TLR9, CD1aþ, CD1bþ, CD1cþ, and CD1dþ cells. Functionally, they exhibit high phagocytic potential and are likely to be rapidly recruited to the site of Ag entry (e.g., mucosal surfaces, skin). They are specialized to be the first to respond to microbial invasion via body surfaces. mDC produce large amounts of IL-12, IL-6, and TNF-a in response to TLR2 and TLR4 ligands, favoring the induction of Th1 responses (17–24). However, the presence of other immunoregulatory molecules, such as prostaglandin E2, may favor mDC priming of Th2 cells. Of note, studies in mice have indicated that conventional DC can be further subdivided on the basis of their expression of CD4 and CD8a in three subsets, of which the CD8aþ CD4 DC appear to be the dominant subpopulation responsible for presentation and cross-presentation of viral Ag (25). On the other hand, pDC are lineage-negative CD11c, CD123hi, CD4þ, CD80þ, CD86þ, CD45RO, CD45RAhi, CD13, CD62Lhi, CD33, CD83, HLA-DRhi, CD206, CCR7þ, TLR2, TLR4, TLR7þ, TLR9þ, CD1a, CD1b, CD1c, and CD1d cells. These DC exhibit plasma cell–like morphology and are functionally characterized by producing large amounts of IFNa and IFN-b in response to TLR9 ligation. These cells are poorly phagocytic, are located mainly in T-cell areas of lymphoid tissues, and are likely specialized to recognize self-Ag or

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blood-borne pathogens. These pDC play an important role in antiviral innate immunity through production of IFN-a and IFN-b. Since blood pDC do not migrate to inflammatory cytokines, they probably reach the LN by responding to SDF-1 (a chemokine expressed in LN) using CXCR4, and CD62L interaction with L-selectin ligands expressed in endothelial venules. This interaction may provide a maturational signal that couples CCR7 with migration, allowing proper positioning of pDC in LN in response to secondary lymphoid tissue chemokines. To add to the complexity of the function of DC in Ag presentation, mDC and pDC regulate each other through the cytokines they release (17,18). Moreover, DC exhibit immunoregulatory effects on B-cell proliferation, differentiation, and isotype switching (26). On the basis of the extraordinary capabilities of DC to prime the immune system, a number of clinical trials have recently explored the use of DC in immunotherapy for cancer on the basis of the injection of Ag-pulsed DC (27). These preliminary studies suggest that this approach is safe and have yielded promising results, however, definitive proof of efficacy is still pending. Further understanding of DC biology is undoubtedly one of the primary areas that, in the coming years, will provide critical information to advance novel vaccination strategies. Other Antigen-Presenting Cells In contrast to DC, the main role of macrophages is to phagocytose, following recognition through PRRs and other receptors such as Ig Fc receptors, and destroy invading microbes. Ag peptides from the pathogens then become available for binding to MHC molecules for presentation to T cells. Once activated, they upregulate their expression of MHC and costimulatory molecules and can become rather effective APC (16,28,29). Nevertheless, macrophages are generally considered to be less efficient than DC at activating naive T and B cells. Of note, macrophages, as well as DC and B cells, have the capacity to present intact Ag on their cell surfaces (29). Macrophages are also a major source of proinflammatory cytokines, including IL-1a, IL-1b, IL-6, IL-8, IL-12, TNF-a, and TNF-b, that exert potent immunoregulatory activities on T-cell responses (28,30). B cells can also function as professional APC by presenting to Th cell peptides derived from soluble Ag following internalization and processing of Ag bound to the B-cell receptor (BCR) complex, which consists of the specific membrane Ig and associated invariants Iga (CD79a) and Igb (CD79b) polypeptides (29,31).

Adaptive Immunity In contrast to innate immunity, the effector mechanisms of adaptive immunity that include, among others, antibodies (Ab) and cell-mediated immune responses (CMI; e.g., CTLs) and cytokines, such as IFN-g and IL-4, are induced following exposure to Ags or infectious agents and increase in magnitude with successive exposures to the specific Ag. This ability to ‘‘recall’’ previous exposures to Ag and to respond rapidly with immunological effector responses of increased magnitude (immunological memory) constitutes the foundation for preventative vaccination against infectious agents. Therefore, this chapter will focus on some of the most significant recent advances in understanding the mechanisms that underlie the development of adaptive immune responses and immunological memory. The main cell types involved in adaptive immune responses, responsible for the recognition of specific Ag, are T and B lymphocytes. However, it is very important to

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emphasize that an adaptive immune response can only be initiated after cells typically considered part of the innate immune response, for example, DC and other APC, uptake, process, and present Ag to naive recirculating lymphocytes in secondary lymphoid tissues (e.g., regional LN). B Lymphocytes, Memory and Antibody Responses to Vaccines Long-term circulating Ab are the correlate of immunity for all human vaccines for which a correlate of immunity is known, and neutralizing Ab are likely the primary functional component of most currently licensed human vaccines (Bacille Calmette-Gue´rin [BCG] and the chicken pox vaccine are the most likely exceptions to this hegemony). Ab circulating in serum or secreted at mucosal surfaces are produced by plasma cells (also known as Ab-secreting cells). Plasma cells are terminally differentiated B lymphocytes, which can arise by differentiation from any of several types of B lymphocytes, including mature naive B lymphocytes and memory B lymphocytes. Therefore, to understand the immunobiology of vaccines and synthesize that understanding during the development of new vaccines, it is critical to understand three components of Ab responses: (i) induction of B lymphocyte responses, (ii) selection of protective Ab, and (iii) generation and maintenance of B lymphocyte memory. These topics are each covered in turn below. Induction of B Lymphocyte Responses B lymphocytes do not produce their effector molecules, that is, Ab, immediately after stimulation; further division and differentiation are required. Pathways and mechanisms of B lymphocyte activation are too complex to cover in detail here (32). Conventional B lymphocyte activation involves two subsequent activation steps: (i) stimulation through the B-cell Ag receptor (surface immunoglobulin, BCR) and (ii) T lymphocyte help. Ag binding the B-cell receptor results in signaling (presuming that the Ag is multivalent and can drive BCR cross-linking/clustering) and internalization of the Ag. The internalized Ag is processed, and peptides derived from the Ag are then loaded onto class II MHC molecules and presented back on the surface of the B lymphocyte. T lymphocyte help is then provided to the B lymphocyte when a CD4 T lymphocyte specific for one of the Ag peptides presented in class II MHC peptide complexes interacts with the B lymphocyte. T lymphocyte help occurs both through integral membrane protein interactions (‘‘costimulatory molecules’’) and secretion of soluble factors (cytokines and related molecules). CD40L (CD154) expressed by CD4 T lymphocytes is the most potent costimulatory molecule for B lymphocyte help and signals to B lymphocytes by binding CD40. The most dramatic evidence of the importance of CD40L-CD40 interactions is the severe Ab deficiency observed in humans with genetic defects in either of these genes (33,34). Inducible costimulator (ICOS) (CD278), which binds CD80 and CD86 expressed by B lymphocytes, is also a potent inducible costimulatory molecule expressed by CD4 T lymphocytes (35–37). IL-21 is a cytokine recently identified as a crucial component of T lymphocyte help, which can be supplemented by other cytokines (38–40). These interactions form the basis of a ‘‘T-dependent’’ (T lymphocyte–dependent) B lymphocyte response. T-independent responses are also possible, but are normally against nonprotein Ag (e.g., polysaccharides) or highly structured polyvalent Ag that potently cross-link BCR (e.g., highly structured viral particles) (41). However, it has recently been shown that there are alternative pathways for B lymphocyte responses that are important in certain circumstances. For

example, B lymphocytes specific for dsDNA can be activated in the absence of T lymphocyte help, and this occurs because of stimulation of B lymphocytes by chromatin through the BCR in combination with stimulation through TLR9, which binds DNA (42). This mechanism is likely generalizable to any Ag that contains a PRR ligand (e.g., dsDNA, LPS) for which the receptor is expressed on B lymphocytes (43). In addition, it has been shown that B-cell-activating factor (BAFF) and related proteins can potently drive B lymphocyte responses by lowering the general activation threshold of the B lymphocytes and reducing apoptosis (44,45). After Ag-specific B lymphocytes have been stimulated to proliferate, some will differentiate into plasmablasts (intermittently differentiated plasma cells that retain the ability to proliferate) and short-lived plasma cells, while others initiate germinal center reactions (46). This decision point is thought to depend on the BCR affinity of a given clone for the Ag. Teleologically, this is thought to reflect a need to rapidly produce functional Ab against an infection to blunt the infection (the short-lived plasma cell response, which is frequently IgM but can be IgG), while also needing to develop a highaffinity Ab response to clear the infection and provide protection from repeat exposures. Memory B lymphocytes are also thought to be biased to plasma cell differentiation after stimulation, which may be directly related to the high-affinity BCR expressed by memory B lymphocytes. Germinal centers are the critical sites for the development of long-term humoral immunity. Memory B lymphocytes are predominantly (or perhaps exclusively) post-germinal center B lymphocytes, as evidenced by high levels of somatic hypermutation in memory B lymphocytes, and the absence of memory B lymphocytes in mice or humans with germinal center defects. Long-lived plasma cells predominantly (or perhaps exclusively) come from post-germinal center B lymphocytes, as evidenced by defects in long-lived plasma cell generation in mice with germinal center defects (47), and the observation that bone marrow plasma cells exhibit DNA and Ab affinity signatures of affinity maturation (48,49). Extensive research has been done into the processes of germinal center B lymphocyte selection and differentiation. It is normally within the germinal centers that affinity maturation occurs. Affinity maturation is the process by which a B lymphocyte clone improves the affinity of its BCR for the cognate Ag through multiple rounds of somatic hypermutation and selection (50). It is known that germinal center B lymphocytes are the only mature cells in the body that undergo somatic hypermutation. Detailed explanations of the various processes, cell types, and genes involved in germinal center reactions are reviewed elsewhere (35,46,51,52). Protective Antibodies While B lymphocytes have roles as Ag-presenting cells, cytokine-producing cells, and in development of lymphoid architecture, the primary purpose of B lymphocytes is to produce Ab. Ab are an indispensable component of protective immunity. But not all Ab are equal. In viral infections, neutralizing Ab—Ab that directly neutralize infectious viral particles—are the primary Ab valuable for protective immunity. The vast majority of neutralizing Ab target proteins that are expressed on the surface of viral particles. In bacterial infections, protective Ab may bind critical Ag on the bacterial surface that are virulence factors (e.g., capsular polysaccharides, LPS, colonization factor fimbriae, or outer membrane proteins) or neutralize secreted bacterial toxins. There are limits to the generalities that

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can be made about protective Ab and their selection, since Ag are unique. However, protective Ab generally function by coating the surface of the pathogen, thereby blocking (or inhibiting) binding to receptors on target cells (53). In the case of bacteria, protective Ab allow destruction of the pathogen by recruitment of complement (bactericidal Ab) or by phagocytosis (opsonophagocytic Ab). Excellent reviews of mechanisms of neutralization are available (53–55). Since neutralizing Ab are extremely detrimental to the survival of pathogens, it is not surprising that viruses and bacteria go to great lengths to avoid or subvert neutralizing Ab responses. Rapid mutation, heavy glycosylation with ‘‘self’’ sugars, inhibition of complement, and exploitation of B lymphocyte repertoire holes are all strategies extensively used by pathogens to avoid neutralizing Ab (53,56–58). The existence of these evasion strategies is also a window into the immunobiology of protective Ab production by B lymphocytes. B-Cell Epitopes In contrast to the epitopes being recognized by T cells (which are composed of amino acid (aa) sequences that are continuous in the primary protein structure), B cells and Ab bind to epitopes that consist of aa that are either continuous or discontinuous (usually associated with lipids or carbohydrates) in the primary protein structure but that are brought together during protein folding. Epitopes composed of continuous and discontinuous aa sequences are referred to as linear and conformational epitopes, respectively (59). Neutralizing Ab often, but not always, recognize conformational epitopes. B Lymphocyte Memory Vaccines are predicated on the existence and function of immunological memory. Therefore, to understand successful vaccine development, it is crucial to understand the nature of B lymphocyte memory. B lymphocyte memory consists of two cell types: memory B lymphocytes and plasma cells. There are several classic examples that have clearly documented longterm protective immunity lasting up to 75 years in humans in the absence of reexposure to the pathogen (60–62), each of which almost certainly reflected long-term B lymphocyte memory. These observations have been crucial in shaping our ideas about immunological memory because they showed that the immune system could remember an encounter that occurred many years ago. The existence of memory B lymphocytes has long been known by observation of anamnestic secondary Ab responses in immunized or previously infected humans or animals who were then reimmunized or reinfected. Immune memory after smallpox vaccination is a valuable benchmark for understanding the kinetics and longevity of memory B lymphocytes in the absence of reexposure to Ag. Immunization against smallpox was standard in the United States and most of the Western world but was stopped in the mid-1970s. The last naturally transmitted case of smallpox disease occurred in Somalia in 1977 and global eradication of smallpox was certified in 1980 (63). Smallpox vaccine-specific memory B lymphocytes can be detected for 60 years or greater after vaccination (64). Importantly, memory B lymphocyte levels appeared to be stable from 10 to 60 years post vaccination, indicating that Ag-specific memory B lymphocytes are maintained by robust mechanisms. There are four models for how human memory B lymphocytes are maintained: (i) Ag-dependent one, (ii) intermittent stimulation by crossreactive Ag, (iii) bystander activation, and (iv) programmed

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homeostatic maintenance. These models are not mutually exclusive and have recently been reviewed elsewhere (46). What is the value of long-term maintenance of memory B lymphocytes? Memory B lymphocytes have several features indicating that they are valuable for protection against infections. First, memory B lymphocytes are present in much greater numbers than naive B lymphocytes of a given Ag specificity (47,65–68). Second, memory B lymphocytes respond to reactivation faster than naive B lymphocytes, require less stimulation, differentiate into plasma cells faster, and have a larger burst size, indicative of resistance to apoptosis (67,69–71). Third, memory B lymphocytes have undergone affinity maturation and therefore produce Ab after reactivation that have substantially higher affinity and/or avidity than Ab produced from naive B lymphocytes. Each of these properties likely makes memory B lymphocytes highly valuable for protection against reinfection, as the memory B lymphocytes are able to make a rapid recall response and produce high levels of highaffinity Ab quickly to limit the spread of the infecting microbe and quell the infection. In situations where Ab are known to be protective but are not present at high enough levels for sterilizing immunity, memory B lymphocytes likely contribute to the observed protection against disease. The hepatitis B virus (HBV) vaccine is a well-characterized example known for Ab titers that drop over several years (72–74), however, many individuals with low or undetectable levels of HBsAg fail to obtain booster immunizations, but nearly of those individuals are still protected from HBV infection (75). Memory B lymphocytes are an appealing explanation for this observation since HBsAg-specific Ab are the correlate of protection for the vaccines, and memory B lymphocytes will rapidly differentiate into anti-HBsAg Ab-secreting cells within three to five days of virus exposure (75–77). The presence of a strong T-memory response might also contribute to this phenomenon. Many human vaccines induce serum Ab responses that persist for decades (78), but it is unclear if there is a long-term decline in the Ab levels, and there are usually nagging questions about the potential of intermittent reexposure to the Ag. One study of long-term Ab levels that attempted to address the reexposure issue was a cross-sectional serosurvey of poliovirus, diphtheria, and tetanus Ab in Sweden (79). Antipoliovirus Ab remained elevated among the various age groups, while tetanus and diphtheria antitoxin titers declined. In other human studies, Ab responses after smallpox immunization (vaccinia virus) can be stably maintained for greater than 60 to 75 years after an initial drop (64,80,81). Data from numerous groups that examined immunity to HBV, tetanus, and diphtheria in humans have shown that anti-HBV, tetanus, and diphtheria serum Ab levels decline substantially over time in vaccinated individuals (72–74,79,82–85). Altogether, these studies show that human Ab can be maintained for greater than 60 years in the absence of reexposure to Ag, but long-term maintenance of Ab levels does not occur in all cases, indicating that not all memory is created or maintained similarly. How is Ab production maintained for decades in some cases? Long-lived plasma cells are crucial for the maintenance of Ab levels. There are currently two main theories for how numbers of Ag-specific long-lived plasma cells are sustained for years after vaccination: (i) competitive/conditional longevity of long-lived plasma cells and (ii) replenishment of longlived plasma cells from differentiating memory B lymphocytes. These models are reviewed and discussed in detail elsewhere (46,86).

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T-Independent B-Cell Responses Production of Ab to most nonprotein Ag, such as glycolipids, nucleic acids, and polymeric polysaccharides, does not require help by cognate T cells and is therefore referred to as thymusindependent (TI) Ag (87–90). In contrast to T-dependent Ag, TI Ag induce mostly IgM Ab of low affinity and, in the majority of cases, do not show significant heavy-chain class switching, affinity maturation, or memory (88). TI Ag have been further subdivided into types 1 and 2 depending on whether they are able (type 1) or not (type 2) to induce immune responses in neonates (88,90). An example of TI-1 Ag is LPS, while most bacterial capsular polysaccharides and carbohydrates are TI-2 Ag. The fact that Ab responses to TI-2 Ag develop later in life is evidenced by the limited responses observed in small infants immunized with, for example, polysaccharide vaccines. In contrast, it is now well established that immunization with conjugate vaccines composed of polysaccharides from, for example, Haemophilus influenzae type B or Salmonella enterica serovar Typhi coupled to T cell–dependent protein Ag elicit strong antipolysaccharide Ab responses that can be increased with repeated immunization and that are very effective in protecting small infants from invasive H. influenzae type B or preschool children from S. enterica serovar Typhi infection (87,91,92). It is unclear what mechanisms underlie these responses and to what extent cytokines derived from APC or small numbers of nonspecific T cells are required to provide a second signal for B-cell triggering after exposure to TI-2 Ag. Because of its importance in vaccine development, particularly for neonates and small infants, this remains an area of intense investigation. T Lymphocytes, Memory and Cell-Mediated Immunity to Vaccines T lymphocytes, in contrast to B cells, recognize peptides (short continuous aa sequences) derived from protein Ag that are presented on the surface of APC in conjunction with class I or class II major histocompatibility complex molecules (pMHC) in the presence of costimulatory molecules (93–98). These Ag may originate from bacteria, viruses, or parasites that have infected host cells and reside intracellularly or from the extracellular environment following internalization by endocytosis (99). Spectacular advances in the ability to track in vivo T cells of known specificity have led to the widely accepted view that naive T cells expressing TCR of the appropriate specificity are activated, almost exclusively, by pMHC complexes presented by DC that have acquired the Ag at the site it entered the host and that, in the presence of the appropriate inflammatory stimuli, migrated to the T-cell areas of secondary lymphoid tissues (e.g., LN, spleen, and Peyer’s Patches [PP]) (98–105). Following Ag presentation and activation in the presence of inflammatory stimuli (e.g., IL-12, IFN-a, IFN-b), T cells undergo an explosive clonal expansion (later to be followed by a contraction phase), mature into effector cells, and migrate to effector sites (98–105). Some Ag-specific T-cell clones remain for long periods of time as memory T cells (Tm) that, upon subsequent exposures to Ag, provide a stronger, rapid, and sometimes qualitatively different specific immune response. Induction of effective Tm cells is critical for successful vaccination. Recent evidence from several laboratories indicates that there are at least two pools of Tm cells: (i) central memory T cells (Tcm) that recirculate through LN and quickly acquire the capacity to produce effector cytokines upon Ag stimulation and (ii) effector memory T cells (Tem) that recirculate through nonlymphoid tissues and are capable of immediate effector function (101,106–108).

There are two main populations of T cells, those expressing CD4 molecules and those expressing CD8 molecules. CD4 and CD8 molecules are T-cell surface glycoproteins that serve as important accessory molecules (coreceptors) during Ag presentation by binding to class II and class I MHC molecules, respectively (93,95,101,106–108). Consequently, CD4 and CD8 molecules, originally used primarily as markers to identify Tcell populations with different functional characteristics, play a major role in class II and I MHC-restricted T-cell activation. CD4þ cells (Th) are mainly involved in inflammatory responses and in providing help for Ab production by B cells, while CD8þ cells, in addition to secreting cytokines, compose the majority of CTLs primarily involved in class I MHC-restricted killing of target cells infected by pathogenic organisms, including bacteria, viruses, and parasites (93,98,99,109,110). Of note, cytotoxic CD4þ T cells have also been described in both animals and humans (111). Cell activation triggered by cross-linking of TCR by pMHC complexes, aided by costimulatory molecules, results in the production of a multitude of molecules with strong immunoregulatory properties collectively known as cytokines and chemokines (discussed below). Acting in concert, cytokines not only modulate the growth, maturation, and differentiation of all cells involved in the generation of adaptive immunity (93,98–100,110) but also strongly regulate innate immunity. Antigen Processing and Presentation to T Cells by Antigen-Presenting Cells An in-depth understanding of the mechanisms involved in these early stages of immune activation is helpful for the development of successful vaccines. Because of space limitations, we will not review here in detail the various pathways of Ag processing and presentation (16). Instead, we will summarize the field and refer the readers to some excellent reviews on this subject. Presentation of Ag to T cells involves a series of intracellular events within the APC, including the generation of antigenic peptide fragments, binding of these peptides to MHC molecules to form stable peptide-MHC complexes, and transport of these complexes to the cell surface where they can be recognized by TCR on the surface of T cells. Two main pathways of Ag processing and presentation (‘‘classical pathways’’), that is, cytosolic and endosomic, have been described (16,112). The ‘‘cytosolic pathway’’ is predominantly used for presentation of peptides produced endogenously in the APC, such as viral proteins, tumor Ag, and self-peptides, associated with class I MHC molecules (59,113–116). The presentation of large numbers of self-peptides complexed to class I MHC molecules results from the inability of APC to differentiate between self and nonself. Under normal conditions, most T cells selected to recognize self-peptides were eliminated during T-cell differentiation or are actively downregulated and, consequently, cannot be activated by self-peptide class I MHC complexes. The second ‘‘classical pathway’’ of Ag processing and presentation, ‘‘endosomal pathway,’’ which is predominantly used for presentation of soluble exogenous Ag bound to class II MHC molecules, involves the capture of Ag by APC, either by binding to a specific receptor or by uptake in the fluid phase by macropinocytosis (117–120). Cross-Presentation and Alternative Pathways for Antigen Processing and Presentation by MHC Molecules Cross-presentation is a process by which Ag is transferred from cells expressing the Ag to host APC (114,115,121,122).

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Historically, this term has been associated with class I–restricted Ag, although cross-presentation can involve either class I– or class II–restricted Ag (122). T-cell activation resulting from cross-presentation can lead to T-cell priming (cross-priming) or T-cell tolerance (cross-tolerance) (122). Cross-priming of naive T cells, largely a function of DC rather than macrophages, has been described for many Ag, including minor histocompatibility Ag, graft tissue Ag, self-Ag, tumor Ag, viral proteins (e.g., Epstein-Barr virus, poliovirus, cytomegalovirus, influenza), ovoalbumin, etc. In addition to the classical pathways of class I and class II Ag presentation, a number of alternative pathways have also been described. For example, (i) an endosome-only route in which Ag is loaded into class I molecules in the endosomes using previously occupied (by peptide exchange) or unoccupied class I MHC molecules, or on the cell surface following the ‘‘regurgitation’’ of peptides processed in vacuolar phagocytic compartments to the cell membrane where they bind surface class I MHC molecules and (ii) a cytosolic route in which Ag are transported from the endosome to the cytosol, allowing them to follow the ‘‘classical’’ pathway (123,124). Several hypotheses have been proposed to explain why cross-priming is a function of DC and not of macrophages, including (i) the fact that the uptake of apoptotic cells is mediated by different receptors in DC and macrophages, (ii) the existence of an endosome-to-cytosol transport mechanism present in DC but not in macrophages, and (iii) the fact that DC are much more efficient in stimulating primary CD8þ CTL responses than macrophages. Whatever the mechanisms involved, an active cross-priming pathway in which DC can process Ag from apoptotic and/or necrotic cells is critical in that it endows these potent APC with the capacity to capture and present Ag from (i) virus-infected, malignant, and transplanted cells that typically lack the accessory functions to be efficient APC and (ii) from pathogens that either do not directly infect APC or suppress their Ag processing and presentation ability following infection. Alternative pathways for Ag processing and presentation have also been described for class II MHC molecules (125). Understanding in depth the mechanisms of classical and alternative pathways of Ag presentation will undoubtedly be important in the design of new vaccine strategies. Nonclassical MHC Class I and Other Molecules Involved in Antigen Presentation In addition to class I and II MHC molecules, a number of other molecules, characterized by their limited polymorphism and lower surface expression, have also been shown to participate in Ag presentation. These include nonclassical MHC class Ib molecules, as well as non-MHC-encoded proteins, such as CD1 and the neonatal Fc receptor (FcRn) (126,127). Class Ib molecules are nonclassical, nonpolymorphic MHC-like molecules that include, in humans, HLA-E, HLA-F, HLA-G, and HLA-H, MIC(MHC class I chain related)A, and MICB (126,127). HLA-E, probably the best characterized of these molecules, plays an important role in NK function. CD1 comprises a family of nonclassical, nonpolymorphic MHC molecules, preferentially expressed by DC and other APC that, on the basis of sequence similarities, have been subdivided into two groups, that is, group 1 (CD1a, CD1b, and CD1c) and group 2 (CD1b) (128). Ag presented by CD1 molecules include microbial lipid, glycolipid, and other nonprotein Ag, such as those present in Mycobacterium tuberculosis and M. leprae (128).

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The Immunological Synapse Studies conducted in the last decade have established that T-cell activation is a complex process that requires at least two signals, one provided by the interaction of the TCR complex (a/b TCR and associated CD3 molecules) with pMHC on APC and a second, complementary signal provided by binding of CD28 (a costimulatory molecule on T cells) to members of the B7 family (e.g., CD80 [B7-1]) on APC (93,94,129). Many additional molecules have also been shown to play important roles in T-cell activation. The term ‘‘immunological synapse’’ has been coined to describe the organized molecular complex that is assembled at the interface between the T cell and the APC where the interaction between the TCR complex and pMHC molecules takes place (93–95,129,130). Formation of immunological synapse (IS) has been described not only for CD4þ and CD8þ T cells but also for NK cells, suggesting that they may be a common feature of lymphocyte activation. The IS has been found to have a remarkable level of organization, characterized by a ‘‘bull’s eye’’ arrangement of supramolecular activation clusters (SMAC) that form within 30 to 60 minutes of T cell– APC contact (94). The central portion of the IS (cSMAC) is enriched for TCR and pMHC complexes, as well as coreceptors CD4 or CD8 and CD28 and its ligand, CD80. The ring around the core (pSMAC) includes other costimulatory molecules (e.g., CD11a/CD18 lymphocyte function associated antigen [LFA]-1) and its ligand, CD54 (intercellular cell adhesion molecule [ICAM]-1), and CD2 and its ligands, CD48 and CD58 (ICAM-3), as well as signaling molecules in the cytoplasmic side of the T cell (e.g., protein kinase C y and the src family kinase lck (93,94)). Interactions among adhesion molecules in the pSMAC, including LFA-1, CD54, CD2, CD48, and CD58, play key roles in maintaining small distances (*15 nm) between apposing T cell and APC membranes and in providing additional signaling (93,131). Important findings derived from the study of the IS include the observations that an intact cystokeleton is an absolute requirement for the T cell but not for the APC and that the formation of a stable IS for at least one hour is required for full T-cell activation. Of note, naive T cells need 20 hours or longer of sustained stimulation to increase their size and become committed to proliferation (130). Several temporal stages have been described in T-cell activation, including T-cell polarization, initial adhesion, IS formation (initial signaling), and IS maturation (sustained signaling) (94). Exposure of naive recirculating T cells to chemokines/cytokines (e.g., signaling from the innate system) and the resulting T-cell polarization, which includes cytoskeletal rearrangements, are required steps that precede the initial APC–T cell interaction and the subsequent IS formation. The precise functions of the mature IS remain controversial. For example, evidence showing that the very early events of TCR signaling occur before the formation of the mature IS suggest that this phenomenon is not an absolute requirement for TCR triggering (132). However, IS appears to play important roles in amplifying weak TCR signaling through concomitant CD28 engagement in the cSMAC. Functional synapses (characterized by the induction of calcium signaling, movement of surface molecules, etc.) between the majority of T cells and DC are formed in the absence of specific Ag, highlighting the uniqueness of T cell– DC interactions (133). Future findings in IS formation in T-cell activation and signaling will undoubtedly lead to new approaches to enhance and/or modulate the type of immune responses induced by vaccination. The formation of an effective IS leads to a complex series of intracellular signaling events that

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result in lymphocyte activation and proliferation. For details on the intracellular signaling pathways and their critical role in the generation of effective adaptive immunity, the reader is referred to a number of excellent reviews (16,94,129,134). Use of Epitopes in Vaccine Development The use of defined epitopes that bind to class I and II MHC molecules (59,135,136) is an attractive vaccine strategy, the advantages of which include (i) the selection of epitopes from conserved regions of various proteins from the microorganism, that is, avoiding variable antigenic epitopes, which can lead to immune evasion, (ii) increased safety, (iii) the ability to select only epitopes likely to play key roles in host defense or tilt the immune response toward desired effector mechanism(s), and (iv) the fact that multiple epitopes can be incorporated in a single vaccine. Moreover, the availability of fluorochromelabeled epitope multimers could greatly aid in the monitoring of immunogenicity in vaccine trials. However, it is unlikely that a vaccine consisting solely of CTL epitopes will be successful. Accumulating evidence indicates that successful subunit peptide vaccines might require the use of the appropriate CTL epitopes in combination with ‘‘universal Th epitopes’’ (i.e., able to bind to a large number of MHC class II molecules) and powerful adjuvants (59,135–137). Moreover, significant efforts are being directed toward enhancing the immunogenicity of subunit vaccines by rationally modifying antigenic determinants (i.e., creating ‘‘agonistic peptides’’) to enhance the host’s immune response through upregulation of Ag recognition (59,135–137). Activation of T Cells by Superantigens Superantigens consist of certain bacterial and viral proteins that, without processing, trigger activation of up to 20% of T cells, including CD4þ and CD8þ cells (138,139). This activation is triggered by high-affinity binding of these superantigens to the lateral sides of class II MHC molecules on APC and to the b chain (Vb) of a/b TCR T cells (138,139). The recognition between T cells and superantigens is specific and clonally variable, since superantigens activate T cells bearing particular Vb regions. Triggering of T cells by superantigens requires the complete TCR-CD3 complex and accessory molecules, including CD4, CD8, CD2, and LFA-1 (aLb2 integrin). Instead of priming an adaptive immune response to the pathogen, T-cell activation by superantigens causes a massive cytokine production and release, mainly by CD4þ T cells. The cytokine response not only causes systemic toxicity but also downregulates the host’s adaptive immunity and might be involved in the triggering of autoimmune diseases. In addition, superantigens result in the activation of APC, leading to the production of proinflammatory cytokines such as IL-1b and TNF-a. The high levels of T-cell and APC cell activation and the ensuing release of cytokines triggered by superantigens play a significant role in the generation of toxic shock syndrome and food poisoning associated with some bacterial infections. Superantigens include products of bacterial and viral origins. Bacterial superantigens include staphylococcal enterotoxins (SE)-A, SE-B, SE-C, SC-D, SC-E, the toxic shock syndrome toxin-1 (TSST-1), and those produced by Streptococcus, Yersinia, and Mycoplasma arthritidis (138,139). Viral superantigens include retroviral glycoproteins, such as the minor lymphocyte stimulating Ag (Mls) produced by the mouse mammary tumor viruses, and products of rabies and moloney leukemia viruses (138,140).

Cytokines, Chemokines, and T-cell Subpopulations: Linking Adaptive and Innate Immunities These immunoregulatory molecules, secreted by immune as well as other cells, play key roles in the clonal expansion of lymphocytes, in mediating the action of effector cells, and in regulating innate immunity (110,141–146). Despite their diversity, most cytokines and chemokines share a number of characteristics, including that (i) they are produced by more than one cell type and act on many different cells (pleiotropism), sometimes exerting more than one effect on a single target cell; (ii) their production follows cell activation, requires de novo RNA and protein synthesis, and is transient; (iii) similar activities are typically performed by more than one cytokine (redundancy); (iv) production of individual cytokines follows the release of other cytokines producing a ‘‘cascading effect;’’ (v) they regulate each other, either positively or negatively, sometimes synergizing or exhibiting additive effects; (vi) they exert their functions by interacting with high-affinity specific receptors on the target cells (109 to 1012 dissociation constant) that they help regulate; (vii) they can exert their activities locally, systemically, or both, by acting in an autocrine (i.e., on the cells that produce them), paracrine (i.e., on adjacent cells), or endocrine (i.e., on distant cells) fashion; and (viii) their actions on the target cells usually involve regulation of proliferation and state of differentiation (110,141–146). The Th1/Th2/Th17 Paradigm Distinct Th cell populations exhibit discrete or overlapping patterns of cytokine production that designate them as Th1, Th2, and Th17 CD4þ T cells (110,141,142,144–150). The predominance of these polarized patterns of cytokine and chemokine production, as well as the temporal sequence of their production, plays a pivotal role in determining the type and characteristics of the effector immune responses generated upon antigenic stimulation, for example, whether the predominant responses will be Ab production (and of which isotypes), enhanced intracellular killing by macrophages, generation of effector CTL, etc., (109,110,144,147). For detailed descriptions of individual cytokines and chemokines, the reader is referred to excellent recent reviews (110,141,142,144–146,149,150). Instead, we will briefly describe how the coordinated induction of chemokines and polarized cytokine patterns plays a role in resistance to disease by invading pathogens and their role in downregulating immune responses. We will also provide some examples on the intricate interactions among these potent mediators in cross-regulating innate and adaptive immunity. Th1 cells are characterized by the production of IFN-g and IL-2, while Th2 cells are characterized by the production of IL-4, IL-13, and IL-33, among others (144,145). Cells producing a combination of these cytokines were named Th0. Moreover, a relatively new subset, named Th17, which appears to play a significant role in autoimmunity and infectious diseases, is characterized by a polarization in the production of IFN-g driven by IL-17 via the intermediate production of IL-23 (143,144,151). Similarly, the function of regulatory T cells (Treg, see below) is characterized by the production of defined cytokines (e.g., IL-10 and TGF-b) (9,144,152,153). CD8þ cells also exhibit type 1 (Tc1) and type 2 (Tc2) cytokine profiles and have been shown to produce IL-17 family cytokines (142,145). Many Th cells exhibit ‘‘mixed’’ cytokine production (e.g., IL-2, IL-4, IL-5, and IFN-g) that does not allow them to be classified into Th1 or Th2 cells, even within populations that are

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polarized toward Th1 or Th2 pattern (154). Moreover, there is a certain plasticity by which Th1 cells can revert to a Th2 phenotype, although reversion in the opposite direction is more difficult (154). A novel subset of T cells able to simultaneously secrete multiple cytokines (named multifunctional T cells) has also been described (155,156). Typically, Th1 responses promote CMI, such as CTL activity, delayed-type hypersensitivity (DTH), antibody-dependent cell-mediated cytotoxicity (ADCC), and macrophage activation, and provide help for the production of certain Ig isotypes (IgG2a in mice, probably IgG2 in humans). Consequently, Th1 responses have been associated with beneficial responses (or found to predominate) in infections caused by protozoa (e. g., Leishmania major, Trypanosoma cruzi), viruses (e.g., influenza), some bacteria (e.g., M. tuberculosis, M. leprae, S. enterica serovar Typhi, Bordetella pertussis, Chlamydia, Listeria monocytogenes), and fungi (e.g., Candida) (157). In contrast, Th1 responses have been associated with detrimental responses in helminthic infections and in pathological conditions such as autoimmune disorders (e.g., experimental autoimmune encephalomyelitis, multiple sclerosis, rheumatoid arthritis), chronic inflammation, transplant rejection, and pregnancy (157). Th2 responses, on the other hand, provide help for Ig production by B cells, including IgE, IgG (IgG1 in mice, IgG4 in humans), and IgA, and promote mast cell and eosinophil growth and differentiation. Accordingly, Th2 responses were found to be associated with beneficial responses in infections caused by helminths (e.g., Trichuris muris, Nippostrongylus brasiliensis, Brugia malayi) and some bacteria (e.g., Borrelia burgdorferi) and with detrimental responses in infections caused by protozoans (e.g., L. major) and viruses (e.g., vaccinia, herpes simplex virus) and in pathological conditions such as allergy and atopic asthma (157). Th1 and Th2 responses are, to a considerable extent, mutually inhibitory phenotypes, leading to the predominance of either Th1 or Th2 responses. Once a T-cell response progresses along a Th1 or Th2 pathway, it tends to become polarized in that particular direction, largely because of the inhibitory effects of Th1 cytokines on Th2 responses and vice versa. For example, IL-10 inhibits cytokine synthesis by Th1 cells and downregulates macrophage activation, while IFN-g inhibits Th2 cell proliferation. In many experimental systems in animals, the resistance or susceptibility to infection in vivo can be altered by modulating the type of cytokine patterns by injection of exogenous cytokines (e.g., IFN-g, TGF-b, IL-12, IL-4) and/or neutralizing monoclonal Ab to cytokines (e.g., anti-IFN-g, antiIL-4), during the early phases of the immune response. Many factors, including the nature and dose of Ag, the route of entry, the nature and maturation stages of the participating DC, the host’s genetic makeup, and the cytokines and chemokines present in the microenvironment during the early stages of lymphocyte activation, are believed to play key roles in determining the predominant polarized cytokine patterns elicited by the invading pathogen (147). For example, it has been suggested that the Th1-promoting capabilities of DC correlate with their ability to produce certain cytokines and chemokines, particularly IL-12, while the absence of these mediators might favor the generation of predominantly Th2 responses (109). In addition to Th1, Th2, and Th17 subsets, studies in human tonsils and in mice have uncovered a population of follicular helper T (Tfh) cells expressing CXCR5 that appears to be critical in the stepwise regulation of the development of Agspecific B-cell immunity in vivo (158). Expression of CXCR5 endows these cells with the ability to reposition into follicular

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regions and germinal centers of secondary LN, increasing the probability of Ag-specific contact between specific Tfh and Agprimed B cells in the draining LN, leading to efficient Ab production (158). In sum, an in-depth understanding of the mechanisms involved in the generation of polarized Th1/Th2/ Th17 and Tfh responses has the potential to significantly impact vaccine development. Multifunctional T Cells Recently, a handful of studies in animals and humans have identified T-cell subpopulations named multifunctional or polyfunctional T cells (155,156). These populations are characterized by the expression of a unique phenotype (CD45R0 CD27intermediate), the simultaneous secretion of several cytokines (chiefly IL-2, IFN-g, TNF-a, and/or MIP-1b) and the ability to degranulate (as measured by CD107 expression) (155,156). These complex responses not only provide additional evidence of the complex functional profiles elicited in CD4þ and CD8þ T cells but it has also been speculated that these cells play a significant role in protection from infection (e.g., smallpox, HIV). These observations have important implications for vaccine development since the induction of multifunctional T cells is an important goal to be achieved in the development of effective vaccines. T Follicular Helper Cells In addition to Th1, Th2, and Th17 subsets, studies in human tonsils and in mice have uncovered a population of Tfh cells expressing CXCR5 that appears to be critical in the stepwise regulation of the development of Ag-specific B-cell immunity in vivo (158). Expression of CXCR5 endows these cells with the ability to reposition into follicular regions and germinal centers of secondary LN, increasing the probability of Ag-specific contact between specific Tfh and Ag-primed B cells in the draining LN, leading to efficient Ab production (158). Regulatory T Cells and Cytokines Involved in the Downregulation of Immune Responses Over the past decade, the existence of several subsets of Treg (formerly known as ‘‘suppressor’’ T cells) exhibiting defined phenotypes and patterns of cytokine production, distinct from Th1 and Th2 cells, has been firmly established (9,152,153). Treg constitute 5% to 15% of CD4þ T cells and are characterized by the expression of the transcription regulator Foxp3, CD25, CD4, and CTL-associated Ag (CTLA)-4, and their growth is dependent on the cytokine IL-2 (9,152,153). The main functions attributed to Treg are to maintain peripheral tolerance by downregulating immunity to self-Ag and limit effector responses to prevent excessive immune-mediated tissue damage. Convincing evidence indicates that Treg decrease the induction of CD4þ and CD8þ immune responses to pathogens and, thus, must be controlled to enable the effective protection against infections and cancer. At least four distinct populations of T regulatory (Tr) cells have been described to date: (i) Tr1 cells, which secrete high amounts of IL-10 and moderate levels of TGF-b, but not IL-2, IL-4, or IFN-g; (ii) Th3 cells, which secrete high levels of TGF-b; (iii) CD4þ CD25þ cells shown to inhibit immunity through undefined mechanisms requiring cell-cell contact; and (iv) CD8þ Treg cells, which can secrete either IL-10 or TGF-b. Because of their predominantly downregulatory properties, production of IL-10 and/or TGF-b is the likely mediator of Treg activity. IL-10 suppresses adaptive immune responses and inflammation while promoting survival

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and differentiation of B cells (159). Similarly, TGF-b inhibits the differentiation of both CD4þ and CD8þ naive T cells into effectors, blocks Th1 and Th2 development by inhibiting transcriptional activators, and downregulates macrophage activation, class II MHC expression, cytokine synthesis, NK cytolytic activities, and activation of neutrophils and endothelial cells by proinflammatory cytokines while promoting IgA production (160). Because of these powerful biological activities, IL-10 and TGF-b are likely to play a dominant role in preventing inappropriate responses to certain self- or environmental Ag. Consensus is emerging that not only the control of Treg function is mediated by cytokines or by stimulation through costimulatory molecules on APC but it also appears that Treg cells can sense pathogens directly through TLR (9,152,153). Other cytokines have also been shown to play a role in downregulating immunity and inflammation. For example, the IL-1R antagonist (IL-1RA), mainly produced by macrophages, neutrophils, keratinocytes, and epithelial cells, acts as a competitive inhibitor of IL-1 by binding to the same receptors as IL-1 without triggering biological function (144). In this way, IL-1RA acts as an anti-inflammatory molecule by blocking IL-1-mediated proinflammatory activities. Soluble cytokine receptors for IL-1, IL-2, and many other cytokines have also been described, and they are postulated to act as anti-inflammatory mediators by binding to the corresponding cytokine in the microenvironment, thereby precluding them from binding to the corresponding cytokine receptor on the surface of the target cells. Given the key role of Treg in the downregulation of immune responses, the understanding of the mechanisms underlying the induction and maintenance of Treg cells, as well as the release of other antiinflammatory cytokines, will be of great importance to further vaccine development against infectious agents and autoimmune diseases, as well as therapies to control graft rejection, inflammation, and allergy. From the above discussion, it is clear that an in-depth understanding of the mechanisms involved in the generation of polarized Th1/Th2/Th17 and Tfh responses, as well as Treg, has the potential to guide vaccine development efforts. A Few Examples of Cytokines and Chemokines Linking Innate and Adaptive Immunities Cytokines and chemokines are the major mediators between APC, lymphocytes, and other cells, and are central to the innate immune system’s capacity to dramatically influence the type and magnitude of adaptive immunity and in the ability of the adaptive immune response to markedly affect inflammatory responses (110,141–146,161,162). For example, cells of the innate immune system (e.g., macrophages, DC) secrete type I IFNs (IFN-a, IFN-b), GM-CSF, IL-1, and TNF-a that promote the activation and differentiation of DC (a major cell type linking innate and adaptive immunity), as well as IL-12, IL-15, and IL-18, which dramatically affect the adaptive immunity induced. Of note, IL-12 has been shown to induce DC and macrophages to secrete IFN-g, a cytokine traditionally considered to be produced by cells of the adaptive immune response (146). In turn, IFN-g markedly affects the activation of cells of innate immunity, for example, by (i) stimulating the cytolytic activity of NK cells, (ii) increasing the expression of class I and II MHC molecules and the production of an array of proinflammatory mediators including IL-1a, IL-1b, TNF-a, IL-6, and IL-8 by APC, and (iii) directly promoting nonspecific killing of bacterial organisms by enhancing the microbicidal activity of macrophages through induction of nitric oxide synthase and protease activity (110,144,161,162).

Because of the central role that cytokines, chemokines, and their receptors play at virtually all levels during the generation of immune responses and as major players in linking innate and adaptive immunity, continued studies on their functions and complex interactions will provide invaluable information to help in the development of novel vaccine strategies.

Models of Memory T-Cell Differentiation In addition to B-cell responses, T cells also play a critical role in vaccine-induced protective immunity. Thus, one of the major goals of vaccination is to induce a pool of long-lived Tm cells that can respond rapidly upon exposure to the pathogen. To design vaccines that will induce long-term T-cell immunity, it is critical to understand how Tm cells are generated. We will now briefly discuss our current understanding of Tm cell differentiation. During the past few years, there has been substantial progress in defining the lineage relationships between naı¨ve, effector, and Tm cells. Several possible models of Tm cell differentiation are shown in Figure 1 (163–165). The first model, the divergent pathway model, is based on the B-cell differentiation paradigm of separate pathways for memory B cells versus plasma cells. While there are some studies supporting this model, there is now compelling evidence that Tm cells are derived from effector cells (165). Thus, it is unlikely that model 1 represents a major pathway of Tm cell generation, and the bulk of the experimental evidence favors the linear differentiation model (model 2). However, this simple linear model does not account for the fact that the majority of effector cells undergo apoptosis and that only a fraction (5–10%) of these cells survive to become Tm cells. A model that incorporates this finding is the dual-effector differentiation pathway (model 3) where one subset of effector cells die and the other subset survives and gives rise to the pool of long-lived Tm cells. A variation of this model is the decreasing potential pathway (model 4) that also incorporates a mechanism for discrimination between effectors that preferentially die and those that survive and differentiate into memory cells. According to this model, the balance between effector cells and memory cells is governed by the duration and level of antigenic stimulation. Cells become more and more terminally differentiated with prolonged antigenic stimulation, and this is accompanied by an increasing susceptibility to apoptosis and a decreasing potential for memory cell development. This model also explains the phenomenon of clonal deletion and T-cell exhaustion that occurs during chronic viral infections with a high Ag load (166). It is also possible to address the development of central and effector Tm cells within model 4. A shorter duration of antigenic stimulation may favor the development of central memory cells, whereas a longer duration may favor effector memory cells. Effector memory cells are characterized by rapid acquisition of effector function and are found predominantly in nonlymphoid tissues, whereas central memory cells are located in lymphoid tissues. This increased understanding of Tm cell differentiation should contribute to the use of a rational approach in designing vaccines to induce highly effective and long-lived T-cell immunity.

Chemokines, Integrins, and Selectins in Cell-Cell Interactions and Lymphocyte Trafficking A highly interrelated network of molecules and their receptors belonging to three separate families has been shown to play a leading role in directing the trafficking of immune cells to sites

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Figure 1 Models of memory T-cell differentiation. Abbreviations: N, naı¨ve cells; E, effector cells; M, memory cells; Tcm, central memory T cells; Tem, effector memory T cells; Tex, functionally exhausted cells; {, cell death. Source: Adapted from Refs. 164 and 165.

of inflammation and secondary lymphoid tissues. These include chemokines, integrins, and selectins. Chemokines Chemokines are a distinct class of cytokines that exhibit chemoattractant properties, that is, they cause cells with the appropriate receptors to migrate toward the chemokine source (149, 167–169). Moreover, chemokines might also regulate the polarity and magnitude of T-cell cytokine responses (149,167). More than 50 chemokines have been identified to date. Most chemokines fall into two main families, cys-X-cys (also called C-X-C; a-chemokines or inflammatory chemokines) and cys-cys (also called C-C or b-chemokines), depending on whether the two Nterminal cystein residues are adjacent or have an additional aa between them. The nomenclature of the ever-growing number of chemokines and their receptors has been revised (149,170). C-X-C chemokines (e.g., CXCL1-CXCL14) are mainly produced by macrophages, neutrophils, fibroblasts, endothelial cells, etc., and attract predominantly neutrophils. In contrast, C-C chemokines (e.g., CCL1–5, CCL7, CCL8, CCL11, and CCL13–27) are mostly produced by activated lymphocytes and attract monocytes, basophils, eosinophils, and lymphocytes (170). Chemokine receptors have also been renamed on the basis of the chemokine subclass specificity of the receptor (e.g., CXCR1–5, CCR1–11) (170). Chemokines and their receptors are now considered to be the most important regulators of leukocyte trafficking. They have been shown to play key roles in many fundamental immunological processes, including, among others, (i) leukocyte binding to endothelium, leading to extravasation (together with selectins and other adhesion molecules; (ii) controlling the traffic of developing B and T cells (through expression of defined sets of chemokine receptors at various

maturational stages, e.g., naive T-cell activation, effector T-cell differentiation, and memory cell development [see below]); (iii) migration of DC to tissues and from tissues to LN (critical for immune surveillance, priming, and tolerance); (iv) migration of monocytes to tissues in response to inflammatory stimuli; (v) interactions between naive T cells and DC and between T and B cells in secondary lymphoid organs; (vi) Th2 attraction of eosinophils (through production of CCL11 [eotaxin-1], CCL24, CCL26, and others that bind CCR3 in eosinophils); (vii) recruitment of Th1 cells to sites of inflammation (through CCR5 and CXCR3 expression); (viii) migration of memory and effector CD4þ and CD8þ T cells to effector sites, such as the gut mucosa (through CCR9) or the skin (through CCR4 and CCR10) in conjunction with other adhesion molecules (e.g., integrin a4b7 for gut-homing and cutaneous lymphoma Ag (CLA) for skin homing [see below]); and (ix) PMN migration and degranulation (through CXCR1 and CXCR2 binding CXCL8 (IL-8)] (149,167–169). Many chemokines are produced spontaneously at particular sites. For example, CCL21 (SLC) and CCL19 (MIP-3b) produced in the T-cell area of LN, attract DC, T cells, and other leukocytes expressing their ligand, CCR7. However, these chemokines are also released by other cells, such as DC, to attract naive T cells. Because of the key role of chemokines in inflammation, several therapeutic modalities directed to control their activity in diseases associated with tissue destruction resulting from inflammatory responses (e.g., allergies, asthma, rheumatoid arthritis, pneumonia) are being vigorously explored. Integrins Integrins are a superfamily of heterodimers consisting of noncovalently associated a and b subunits that, by mediating

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cell-cell (e.g., endothelial cells) and cell-matrix (e.g., collagen, fibronectin) adhesion, play a major role in T-cell activation and homing to secondary immunological organs and sites of inflammation (171). The fact that most members of the integrin family involve a common b subunit associating with specific a subunits leads to the original classification of integrins into the so-called b-integrin families. A total of 8 b subunits and 18 a subunits have been identified to date (171). In some cases, a particular a chain subunit can bind to more than one b chain. Among the key integrin families involved in lymphocyte homing are members of the b1 (CD29) family, which consists of at least nine members (heterodimers composed of b1 and a1/a8 or aV), the b2 (CD18) family (heterodimers composed of b2 and aL, aM, or aX), and the b7 family (heterodimers composed of b7 and a4 or aHML [Human Mucosal Lymphocyte Antigen]). The family of b2 integrins, which is expressed on all leukocytes and has been shown to mediate transmembrane signal transduction, includes three homologous heterodimers composed of a common b2 chain (CD18): complement receptor 3 (CR3, CD11b/CD18, found predominantly in monocytes, NK, and neutrophils, as well as in some lymphocytes), CR4 (CD11c/CD18, found in macrophages, granulocytes, and some T cells), and aLb2 integrin (LFA-1, CD11a/CD18) that is expressed in lymphocytes, monocytes, NK, and other leukocytes (172). CR3, for example, recognizes the iC3b complement component, ICAM-1, fibrinogen, and other ligands and relays this ‘‘proinflammatory information’’ to the cytoplasm via exodomain interactions (172). Selectins Selectins comprise a family of three carbohydrate-binding molecules involved in leukocyte-endothelial cell adhesion: L-selectin (CD62L; present in T and B lymphocytes, monocytes, NK, neutrophils, and other cells), E-selectin (CD62-E, ELAM-1; present in endothelial cells), and P-selectin (CD62-P; present in platelets, activated endothelial cells, and megakaryocytes). CD62L (peripheral LN homing receptor) is expressed at high levels in most naive CD45RAhi CD45ROlo T cells, and its expression declines after activation. Thus, CD62L is expressed at low levels in most Tm cells. However, it is important to note that evidence in both rodents and humans indicates that some Tm cells are capable of reexpressing CD62L. CD62L plays a key role in the interaction of lymphocytes with high endothelial venules (HEV) by recognizing carbohydrate moieties in the surface glycoproteins of endothelial cells, including glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), CD34, and peripheral node addressin (PNAd) in LN (173,174). Integrins, Selectins, and Chemokines: An Integrated View of Lymphocyte Trafficking Since the first step in lymphocyte migration to peripheral tissues involves leukocyte adhesion to the vascular endothelium, the capacity of integrins to bind to vascular addressins plays a critical role in lymphocyte homing. For example, integrins belonging to the b7 integrin family, such as a4/b7 (lymphocyte Peyer’s patch adhesion molecule [LPAM]-1) and aE/b7 (HML-1) appear to be critical for lymphocyte homing to mucosal tissues (102,169,174). The a4/b7 integrin binds to the mucosal addressin cell adhesion molecule 1 (MadCAM-1) present in endothelial cells of HEV (102,169,174). HEV, present in LN, PP in the intestine, tonsils, adenoids, appendix, and aggregates of lymphoid tissues in the gut mucosa, as well as in

chronically inflamed nonlymphoid tissues but not in spleen, are critical target sites for lymphocyte recirculation (102,174). The aE/b7 (HML-1) integrin, which binds to the E-cadherin chain expressed by mucosal epithelial cells, appears to be important in lymphocyte homing to the gut epithelium (169). Another integrin, a4/b1 (CD49d/CD29, very late antigen-4 [VLA-4], LPAM-2), which binds to the vascular ligand vascular cell adhesion molecule 1 (VCAM-1) that is expressed primarily on the endothelium of nonmucosal sites of inflammation, appears to play a key role in homing of activated T cells to nonmucosal sites. Similarly, LFA-1 (aLb2 integrin) is also involved in homing of activated lymphocyte to peripheral tissues by binding to ICAM-1 and ICAM-2 present in HEV (102). Differential expression of integrins (e.g., a6b1 in Th1 cells) can also contribute to the distinct homing behavior of Th1 and Th2 cells (173). Taken together, the information discussed above on the role of chemokines, integrins, and selectins in lymphocyte trafficking clearly demonstrates that the homing potential of immune cells depends on the coordinated production of multiple molecules and expression of the appropriate receptors. For example, the fact that CD62L expression declines markedly after lymphocyte activation, which occurs concomitantly with the increased expression and affinity of adhesion molecules such as a4/b7 integrin, a4/b1 integrin, and LFA-1 (aLb2 integrin), promotes activated T cells to leave the LN and migrate to sites of inflammation in peripheral tissues, including the gut-associated mucosa. In addition, concomitant expression of other receptors (e.g., CCR9 in a4b7hi cells), might help direct their homing to particular areas of the mucosa, for example, the intestinal mucosa, which has been shown to produce thymusexpressed chemokine (TECK), a CCR9 ligand (102,169). TECK is absent or weakly expressed in other segments of the gastrointestinal tract (e.g., stomach and colon), and only a portion of lymphocytes in the colon expresses CCR9. Another example of the complexity of the signals involved in lymphocyte homing is trafficking to the skin. Lymphocytes with skin homing potential express CLA but do not express integrin a4b7. These lymphocytes concomitantly express CCR4 (the ligand for CCL17, produced by cutaneous endothelium) and/or CCR10 (the ligand for CCL27, produced by keratinocytes) (169). Elucidation of the molecular basis of lymphocyte homing will undoubtedly have an enormous impact on the vaccine development field in years to come. This knowledge will help determine the most appropriate routes of immunization or means to target Ag to the correct site for optimal presentation depending on the desired effector immune response. It will also help predict the most likely site and type of effector immunity elicited by immunization.

FUTURE DIRECTIONS Throughout this chapter, largely because of space constrains, we focused on only a handful of key areas during the generation of effector and memory cells that hold great potential for advancing the development of safe and effective vaccines. These topics are by no means exclusive of many others such as Ag-Ab interactions, B- and T-cell development and differentiation, Ig class switching, organizational structure of lymphoid organs and tissues, immunological tolerance, immunosenescence, immune responses in neonates, etc., that are also important to vaccine development and are covered in other chapters in this book.

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In coming years, the increasing use of the technologies described above (e.g., genomics, proteomics, systems biology) should advance our understanding of the mechanisms underlying the induction, effector and memory phases of the immune response, and the tactics and molecular processes utilized by the immune system to protect the host against invading pathogens. The areas most likely to be pivotal in the development of vaccines include (i) in-depth understanding of the pathways and antigenic epitopes involved in class I and class II MHCrestricted immune responses, (ii) further delineation of the function of DC cell subsets in linking the innate and immune system, (iii) the precise role of costimulatory and adhesion molecules in Ag presentation and homing of effector and memory lymphocytes, (iv) better understanding of the rules governing the generation of cells secreting polarized cytokine profiles, (v) further characterization of Treg and Tfh, (vi) novel cytokine- and chemokine-mediated immunoregulatory mechanisms, and (vii) generation and maintenance of large pools of memory B and T lymphocytes. These advances should create opportunities to modify Ag structure and vaccine formulations and targeting, leading to more efficient immune induction, more persistent immune responses, and enhanced memory. For example, high hopes are held for targeting Ag to APC in conjunction with immunoregulatory cytokines and on the effective use of adjuvants. One goal is to develop vaccines that are ‘‘better than nature’’ in the sense that they may induce immune responses that are superior to natural infections in generating protective immunity.

ACKNOWLEDGMENTS This work was supported in part by Grants R01-AI036525 and R01-AI057927 and Research Contract N01-AI30028 (to M.B.S.) from the National Institutes of Health.

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18 Modulating Vaccine Responses with Innate Immunity Bali Pulendran Department of Pathology, Emory Vaccine Center, Atlanta, Georgia, U.S.A.

Jeff Powell VaxInnate Corporation, New Haven, Connecticut, U.S.A.

Richard A. Flavell Department of Immunobiology, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, Connecticut, U.S.A.

INTRODUCTION Vaccines can be divided into two major families—live, attenuated vaccines and nonreplicating vaccines. Live, attenuated vaccines, such as the measles, rubella, oral polio, and oral typhoid vaccines, consist of a weakened version of the pathogen, which is not pathogenic but immunogenic. The nonreplicating vaccines usually consist of components of the pathogen, isolated chemically or through recombinant DNA technology (e.g., ‘‘subunit vaccines’’ such as the Hepatitis B vaccine). A central component of subunit vaccines are additives called adjuvants, which enhance the magnitude of immune responses. Currently, only alum—an aluminum salt–based substance—is licensed for clinical use in the United States. Live vaccines contain their own ‘‘adjuvants’’—microbial or viral stimuli that activate the immune system. Despite the critical importance of adjuvants in generating robust immune responses, we are largely ignorant of how they work. Clearly, mechanistic insights into how successful vaccines and adjuvants mediate robust and long-lived protective immunity would be of great value in the design of future vaccines against global pandemics and emerging infections. In this context, recent advances in innate immunity research are beginning to provide new insights. It is now clear that many microbial stimuli, including components of vaccines, act via toll-like receptors (TLRs), which therefore represent promising therapeutic targets for the development of novel adjuvants. However, recent evidence suggests that some adjuvants can induce robust adaptive immunity in a TLR-independent manner, perhaps through other receptors in the innate immune system. Therefore, understanding the precise roles played by TLRs and other nonTLRs in the induction and regulation of adaptive immune responses is critical for the design of optimally effective vaccines. In this chapter, we review emerging advances in innate immunity, and how they impact our understanding the mode of action of many successful vaccines and adjuvants and guide the design of future vaccines.

The New Science of Adjuvants The ‘‘innate’’ immune response is an evolutionarily ancient system of host defense, which occurs within minutes or hours

of vaccination, or pathogen entry (1). A critical cell type in the innate system is the dendritic cell (DC), which has evolved to ‘‘sense’’ components of microbes or viruses, to process this information, and then to convey instructive signals to antigenspecific T and B lymphocytes in the adaptive immune system (2). There are several different mechanisms by which DCs can sense microbial stimuli contained in pathogens or vaccines. A direct mechanism of sensing components of bacteria, viruses, parasites, or fungi is with so-called pattern recognition receptors (PRRs), which are expressed on the surface or within intracellular compartments of DCs (3). A second method is an indirect mechanism, in which DCs do not sense structural components of microbes directly but rather perturbations in the local cellular microenvironment caused by the infection or vaccination. There are several different subpopulations of DCs that differ in their surface phenotype, function, and immune stimulatory potentials. These will be discussed in detail later (4,5). Direct Sensing of Microbes and Vaccines Toll-like receptors. The innate immune system can recognize microbes directly through various innate immune receptors expressed by DCs. Preeminent amongst these are the TLRs, of which at least 11 have been described and which sense a wide array of stimuli. TLRs constitute an evolutionarily conserved family of receptors, called the interleukin (IL)-1R/ TLR superfamily, and are widely expressed on a variety of innate immune cells, including DCs, macrophages, mast cells, neutrophils, and epithelial and endothelial cells. TLRs have broad specificity for conserved molecular patterns shared by bacteria, viruses, and parasites, respectively (Fig. 1) (6,7). Moreover, different TLRs are expressed by distinct subpopulations of DCs and in distinct cellular compartments. TLR4, for example, is expressed on the surface membrane of human myeloid DCs and monocytes, and is essential for the recognition of lipopolysaccharide (LPS) (7). TLR2 heterodimerizes with TLR1 or TLR6, is expressed on human myeloid DCs, and senses a wide range of stimuli including cell wall components of gram-positive bacteria, such as peptidoglycans, lipoproteins and lipoteichoic acids, and the fungal cell wall zymosan (reviewed in Refs. 6,8). In contrast, TLR9 and TLR7 are

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Figure 1 Some innate immune receptors, and their ligands and intracellular signaling pathways. The TLRs consist of at least 11 receptors, some of which are expressed on the surface and the others in endosomal compartments of DCs. Most TLRs signal via an adapter protein called MyD88, but other adapter proteins such as TRIF (TLR3 and TLR4), TRAM (TLR4), and TIRAP (TLR2) also mediate signals via specific TLRs. CLRs consist of more than 20 extracellular receptors, which bind a wide array of microbial and viral stimuli. The signaling pathways that mediate their effects are poorly understood. NLRs (NOD protein-like receptors) consist of more than 20 cytosolic proteins that are thought to be involved in the recognition of a variety of microbial stimuli. Ligand recognition by NOD2 results in the recruitment of RICK/RIP2 that results in NF-kB activation, and other NLRs regulate the cysteine protease caspase-1, within a dynamic multi-protein complex called the ‘‘inflammasome,’’ leading to the cleavage and activation of pro-inflammatory cytokines such as IL-1b and IL-18, as well as host-cell death. RLRs, which include RIG-I and MDA5, are intracellular receptors that sense double-stranded RNA and mediate antiviral responses. Abbreviations: TLR, toll-like receptor; NOD, nucleotide-binding oligomerization domain protein.

expressed in the endosomes of plasmacytoid DCs (pDCs) and are respectively involved in the recognition of viral and intracellular bacterial DNA, and single-stranded RNA (reviewed in Ref. 6). TLR5 is expressed predominantly on intestinal epithelial cells and antigen-presenting cells, and recognizes flagellin (9). C-type lectin-like receptors. Although much research has focused on the TLR family as innate sensing receptors, emerging evidence suggests that other receptor families may also contribute to the innate immune response. In mammals, the C-type lectins represent a conserved family of receptors, which contain one or more C-type lectin domains and are expressed as transmembrane receptors on myeloid cells (10,11). Many C-type lectins are ‘‘orphan’’ receptors, for which no ligand has yet been described. C-type lectins bind

carbohydrates, not only from pathogens, but also from selfglycoproteins, and play an important role in cell adhesion, migration, and phagocytosis. DCs express several C-type lectins, such as the mannose receptor, DEC205, DC-specific ICAM-grabbing nonintegrin (DC-SIGN), blood dendritic cell antigen 2 (BDCA2), dectin-1, DC immunoreceptor, DCassociated lectin 1, C-type lectin receptor 1, Langerhans cells (LC)-specific C-type lectin (langerin), and DC–asialoglycoprotein receptor (DC-ASGPR)/macrophage galactose N-acetylgalactosamine-specific lectin 1 (10,11). Many C-type lectins have been shown to mediate cytokine responses in macrophages and DCs in response to a variety of microbial stimuli. For example, DC-SIGN can recognize a host of microbial components such as glycoproteins of HIV and HCV, and

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carbohydrates of Helicobacter pylori and Mycobacterium tuberculosis (10,11); dectin-1 recognizes and mediates signal transduction of DCs and macrophages in response to the yeast cell wall zymosan (12).

Nucleotide-binding oligomerization domain protein-like receptors. In mammals, the NLR family consists of more than 20 cytosolic proteins, which are thought to be involved in innate recognition of intracellular bacteria and the induction of inflammatory responses. NLRs contain a C-terminal leucinerich repeat (LRR) domain, a central nucleotide-binding NACHT domain, and an N-terminal protein-protein interaction domain usually composed of a caspase activation and recruitment domain (CARD) and/or a pyrin domain (13–15). Several NLRs have been implicated in innate recognition of intracellular bacteria and the induction of the NF-kB signaling pathway (13–15). Ligand recognition by nucleotide-binding oligomerization domain containing 2 (NOD2) results in the recruitment of RICK/RIP2 that leads to NF-kB activation (16). In addition, it has been proposed that other members of this family of cytosolic proteins regulate the cysteine protease caspase-1 within a dynamic multiprotein complex called the ‘‘inflammasome,’’ leading to the cleavage and activation of proinflammatory cytokines such as IL-1b and IL-18 (17–19), as well as hostcell death. Thus, NALP1 and NALP2/3 represent NLRs that form two major types of inflammasomes. NALP3 is known to be activated by several stimuli including bacteria such as Francisella, Listeria, and Legionella (20), bacterial RNA, antiviral compounds, and endogenous danger signals such as ATP (recognized via the P2A7 receptor and presumably released by dying cells) and uric acid crystals (21,22). The physiological stimulus for NALP1 is unknown, although cell rupture is thought to be important. IPAF is another NLR involved in the recognition of Salmonella and Pseudomonas, which also induces caspase-1 activation (23,24). In addition, NAIP5 is also known to be involved in the detection of Salmonella flagellin in the cytoplasm (25). Exactly how TLR5, IPAF, and NAIP5 contribute to the adjuvant effects of flagellin remains to be determined. Mutations in certain nucleotide-binding oligomerization domain (NOD) genes have been associated with inflammatory disorders or immunodeficiency in humans. Thus, NOD2 is involved in the predisposition to Crohn’s disease and Blau syndrome, and intriguingly, NOD2 appears to act as intracellular sensor of muramyl dipeptide (MDP), a component of bacterial peptidoglycan (13–15). Indeed, MDP acts as an adjuvant in a NOD2-dependent manner (26). Together, these findings suggest that NOD proteins may play a role in the recognition of intracellular bacteria and perhaps in the regulation of immune responses in vivo. RIG-I-like receptors. Infection of a cell with RNA virus generates double-stranded RNA, which is recognized by RIG-I, a cytoplasmic protein with a helicase and caspase-recruiting domain (CARD) that mediates type I interferon (IFN) production (27,28, reviewed in Ref. 6). Melanoma differentiation–associated gene 5 (MDA5) is a homologous cytosolic protein that also recognizes double-stranded RNA and mediates transcription of type I IFN (8,28, reviewed in Ref. 6). RIG-I and MDA5 recognize different RNA viruses—cells deficient in RIG-I show impaired type I IFN responses to paramyxoviruses, influenza virus, and Japanese encephalitis virus, while MDA5-deficient cells respond poorly to picornaviruses. Sensing double-stranded viral RNA is also thought to be mediated by MDA5. Recent studies have identified a mitochondrial adapter involved in RIG-I- and MDA5-mediated type I IFN induction [called IFN-b

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promoter stimulator 1 (IPS-1) (reviewed in Ref. 6)], mitochondrial antiviral signaling protein (MAVS), or virus-induced signaling adapter (VISA) (reviewed in Ref. 6). Sensing systems also exist for DNA viruses and are under intensive study. Indirect Sensing of Microbes and Vaccines In the first few seconds or hours following infection or vaccination, several different cell types respond. Macrophages, natural killer (NK) cells, NK T cells, mast cells, basophils, stromal cells, epithelial cells, and endothelial cells also express PRRs and are thus able to sense microbial stimuli directly and secrete inflammatory mediators. These inflammatory mediators, which include cytokines, heat-shock proteins, and uric acid crystals can be sensed by DCs. Importantly, therefore, DCs are equipped with multiple surveillance mechanisms that can sense pathogens either directly or indirectly and thus represent an important nodal point in which pathogen- or vaccineassociated signals are integrated and transmitted to the adaptive immune system.

Modulation of Adaptive Immunity by Innate Immunity Induction of long-lived plasma cells that secrete neutralizing antibodies, and persistent antigen-specific CD4þ and CD8þ T cells, and the quality of the T-helper response are important correlates of protective immunity against several pathogens. For example, effective immunity against many viruses and intracellular bacteria require Th1 cells that secrete IFN-g and which favors cytotoxic T cells (CTLs); in contrast, Th2 cells secrete IL-4, IL-5, and IL-13, which induce immunoglobulin (Ig)E and eosinophil-mediated destruction of helminths. The recently discovered Th17 cells secrete IL-17 tumor necrosis factor (TNF)-a and IL-22 and likely play a role in controlling infections caused by fungi and extracellular bacteria (29). Finally, T regulatory cells express the transcription factor Foxp3, secrete transforming growth factor (TGF)-b and IL-10, and suppress the activation of effector T cells as well as innate immune cells. Given these functional specializations, learning how to stimulate the appropriate type of T-cell response is crucial to inducing optimally effective immune responses against different infections. It is now clear that the innate immune system plays a fundamental role in determining the magnitude and quality of the adaptive immune response. Therefore, knowledge of which innate immune receptors and cell types to trigger to stimulate a given type of response is of critical importance in the design of vaccines. How Dendritic Cells Modulate Adaptive Immunity DCs are very efficient antigen-presenting cells, and there is evidence that they are essential for the priming of naı¨ve CD8 T cells to various pathogens (2). DCs, in fact, consist of a network of distinct subpopulations, which differ in their phenotype, microenvironmental localization, and functions (4). The existence of such DC subsets represents one level by which DCs control immune responses. Thus, distinct DC subsets can differentially modulate T-helper responses (3,4,30) and differ in their capacity to present antigen to CD8þ T cells. However, microbial stimuli and local microenvironmental milieu can also influence the T-helper response (3,4,30). For example, in mice splenic CD8aþ versus CD8a DCs have intrinsic propensities to stimulate Th1 versus Th2 responses, respectively. In humans, pDCs can be induced by certain viruses to secrete IFN-a and

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Interestingly, Th1- or Th2-promoting stimuli differentially induce Delta versus Jagged (two Notch ligand families), which act as instructive signals for Th1-versus-Th2 differentiation (31). In addition to microbial stimuli, the cytokine and chemokine milieu in the local microenvironment can also modulate DC function and immune responses. For example, DCs from the Peyer’s patches preferentially prime Th2 responses, while total spleen DCs preferentially prime Th1/Th0 responses (32).

differentiate into Th1-inducing DCs (3), whereas IL-3 induces such DCs to differentiate into Th2-inducing DCs (3). Furthermore, distinct DC subsets are also differentially equipped to present antigens to CD8þ T cells. The acquisition of exogenous antigens and their entry into the major histocompatibility complex (MHC) class I–restricted antigen presentation pathway are necessary for stimulation of antigen-specific CD8þ T cells. This process is called ‘‘cross-presentation’’ or ‘‘cross-priming,’’ and distinct DC subsets appear to have intrinsic propensities to cross-present antigens. For example, in mice, the CD8aþ DCs that can be induced to secrete higher levels of IL-12 and prime Th1 responses are also more efficient at cross-presentation of antigens to CD8þ T cells (reviewed in Refs. 3,4). In contrast, CD8a DCs, which prime Th2 responses, are more efficient at presenting antigens to CD4þ T cells (3,4). These differences are consistent with the differential expression of proteins involved in processing of antigen into the class I or class II pathways, respectively. Despite the functional specializations of DC subsets, they also exhibit a great degree of ‘‘plasticity’’ in their function, especially if the DCs are immature and not yet fully differentiated. Thus, their intrinsic properties can be modified by microbial stimuli (3–5). For example, different forms of the fungus Candida albicans provide distinct instructive signals to guide DC differentiation into Th1- or Th2-inducing cells; Escherichia coli LPS induces IL-12p70 in DCs, which elicits Th1 responses, while Porphyromonas gingivalis LPS, schistosome egg antigens (SEAs), filarial nematode–secreted products, or cholera toxin fail to induce IL-12p70 and stimulate Th2-like responses (reviewed in Refs. 3,4,30).

How Toll-like Receptors Modulate Adaptive Immunity The nature of the PRRs that mediate DC activation, and the immunological consequences of signaling via such PRRs are under intense study. Stimulation of DCs with TLR ligands results in their activation and the secretion of proinflammatory cytokines. There is now overwhelming evidence that TLR ligands can induce robust antigen-specific T- and B-cell responses in vivo (33,34). Several TLR ligands are under development as candidate adjuvants or components of vaccine formulations (Table 1). Initial studies suggested that all TLRs induced a similar signaling pathway and biological responses from DCs. However, subsequent studies show that triggering different TLRs results in distinct but overlapping signaling pathways and biological responses. A canonical signaling pathway of TLR activation consists of the recruitment and subsequent phosphorylation of IL-1R1-associated protein kinases 1 and 4 (IRAK 1–4) to the TLR complex. This results in association with the TNF receptor– associated factor 6 (TRAF 6), leading to the activation of the

Table 1 Innate Immune Activation by Some Licensed Vaccines and Vaccine/Adjuvant Combinations, and Emerging Adjuvants Being Used in Combination with Various Vaccines Innate immune mechanism

Type of immune response

Smallpox (Vaccinia virus)

Inhibits DC activation and causes cell death52 Blocks TLR4 and TLR3 signaling53

CTLs Neutralizing Ab

Yellow Fever vaccine YF-17D

Activates multiple DC subsets through TLRs 2,7,8 and 948 Activates RIG-I and MDA-5(67)

CTLs Th1/Th2 Neutralizing Ab

BCG

Activates TLR2, 4, 9 and DC-SIGN49–51

Th1/Th2

Alum

TLR signaling not critical for induction of Ab responses56 Induces caspase-1/inflammasome activation in DCs57

Th2 Antibody

MF59 (licensed in Europe in combination with Novartis influenza vaccine).

Mechanism unknown. Enhanced uptake by antigen presenting cells probably important

Th2 Antibody

AS04 (MPL derivative; licensed for use in Europe in combination with Hepatitis B vaccine)

TLR4 agonist59,61

Th1 Antibody

CpG DNA

TLR9 ligand60,61

Th1, Antibody

TLR7/8 ligands

TLR 7 ligands63,64

Licensed Vaccines

Licensed Adjuvant/Vaccine Combinations

Some emerging adjuvants that stimulate innate immunity in clinical trials in combination with a variety of vaccines

9

Flagellin-protein fusions

Activates TLR5 , IPAF and NAIP5

Cationic-DNA lipid complexes

Not known62

Th1, Antibody 23–25

Th1/Th2 Th1, CTL

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NF-kB and mitogen activated protein kinase (MAPK) signaling pathways, which mediate the induction of certain inflammatory cytokines such as TNF-a and IL-6 (6). Despite the common signaling pathways induced by different TLRs, emerging evidence suggests that different TLRs can also mediate distinct signaling pathways and biological responses. For example, while most TLRs induce strong IL-12p70 from DCs, which subsequently stimulate Th1 responses (35), only a subset of TLRs can mediate the induction of type I IFNs and Th2 cytokines. Thus, TLR3, TLR4, TLR7, and TLR9 can induce type I IFNs, which are important for antiviral defense (reviewed in Refs. 3,4,30). Furthermore, there is evidence that TLR ligands can also stimulate Th2 or T regulatory responses (reviewed in Refs. 3,4,30). The molecular mechanism by which specific TLR ligands favor a Th2 bias remains to be established, although recent work suggests that the robust and sustained phosphorylation of extracellular signal-regulated kinase (ERK) mitogen activated protein (MAP) kinase results in phosphorylation of the AP-1 transcription factor c-Fos in DCs, which in turn suppresses expression of the Th1-defining cytokine IL-12, thus favoring a Th2 bias (reviewed in Refs. 3,4,30). In addition to their effects on Th1/Th2 modulation, signaling via TLR3, TLR7, and TLR9 has been shown to enhance cross-presentation by DCs, although the molecular mechanism of this is not understood (reviewed in Refs. 3,4,30). Finally, emerging evidence suggests that TLRs also regulate B-cell responses. Injections of TLR ligands are known to stimulate robust antibody responses in mice and humans. Interestingly, it appears that direct activation of B cells by TLR ligands is important in mediating critical aspects of the B-cell response. For example, a recent study demonstrates that generation of Tdependent antigen-specific responses requires activation of TLRs in B cells (34). How Non-Toll-Like Receptors Modulate Adaptive Immunity In contrast to our knowledge of how TLRs modulate adaptive immune responses, there is still scant knowledge of what effects other families of PRRs exert on adaptive immune responses. RIG-I-like receptors (RLRs) are present in the cytoplasm where they detect viral dsRNA or ssRNA and mediate antiviral type I IFN responses, but their roles in modulating adaptive immune responses are not known. In the case of C-type lectin-like receptors (CLRs), specific details of their roles in adaptive response are still sketchy, but emerging evidence suggests an important role for them in regulating the T-helper balance. For example, H. pylori can modulate the Th1/Th2 balance through phase-variable interaction between lipopolysaccharide and DC-SIGN (36). The major glycoprotein allergen from Arachis hypogaea, Ara h 1, is a ligand of DC-SIGN and acts as a Th2 adjuvant in vitro (37). Mice lacking SIGN-R1 have enhanced Th1 responses and diminished Th2 responses (38). The fungal cell wall zymosan, which signals through TLR2 and dectin-1, stimulates DCs to produce robust levels of IL-10, little or no IL-12p70, and IL-23, and induces a mixed T-helper response consisting of both Th17 cells and IL-10þIFN-g IL-4 Tr1 cells (39). Finally, with regards to NLRs, there is little understanding of how they contribute to the induction and modulation of adaptive immune responses. However, several studies performed over the past 20 years with purified and synthetic components of peptideoglycan, including MDP, have demonstrated induction of potent immunity against bacterial, fungal, parasitic, and viral infections (40,41). In fact, MDP in certain

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formulations was tested as a potential vaccine adjuvant by scientists at Syntex in the mid-1980s, but its pyrogenic effects precluded further development (42). The study of mice deficient in NOD2 has yielded conflicting results. Strober and colleagues reported that NOD2 is a negative regulator of TLR2-mediated NF-kB signaling, and IL-12 and Th1 responses (43). However, this is not consistent with other groups that failed to find an effect on IL-12 secretion induced by TLR2 ligands (26,44). The possible influence of other NLRs such as NALP1, NALP2, NALP3, NALP12, IPAF, and NAIP5 on the adaptive immune response is at present unknown.

From Innate Immunity to Vaccines Live, Attenuated Vaccines Vaccines represent one of the most cost-effective public health tools in history, yet most of the successful vaccines have been made empirically, including a number of live vaccines. Smallpox vaccine, which has been administered to over a billion people and eradicated the disease worldwide, consists of live vaccinia virus (attenuated for humans by means of host restriction) (45). Live, attenuated yellow fever vaccine strain 17D (YF-17D) is considered to be one of the most effective vaccines and has been administered to nearly half a billion people (46). A single injection of this vaccine can induce a broad spectrum of adaptive immune responses, including a mixed Th1/Th2 profile, robust CD8þ T-cell responses, and neutralizing antibody responses that last for up to 30 years after vaccination. Despite these successes, we do not have a mechanistic understanding of how such successful early vaccines derived empirically work. Since attenuated vaccines consist of viruses (e.g., measles, yellow fever) or bacteria [e.g., bacillus Calmette-Gue´rin (BCG), Ty21a], it is very likely that they signal through several different innate immune receptors, including TLRs. However, the nature of these receptors and how signaling through several such receptors might be integrated in DCs to result in a given type of immune response is not known. In this context, a recent study suggests that activating DCs via different TLRs may result in a synergistic activation of specific genes in DCs (47). Therefore, whether combination of TLR ligands could result in synergistic activation of adaptive immunity remains to be determined. Importantly, persistent production of neutralizing antibody by long-lived plasma cells is a cardinal correlate of protective immunity for many vaccines. Yet we have no knowledge of the innate immune signals that would stimulate such a response. Such knowledge should be useful in guiding the design of future vaccines in terms of what ‘‘innate immune buttons’’ to push, to elicit a particular type of adaptive immune response. Moreover, it would obviate the need to use live vectors, which have been associated with rare, albeit very serious, adverse reactions. Therefore, a better understanding of the mode of action of empirical vaccines should permit the rational design of safer vaccines that recapitulate the immunogenicity of our best vaccines. In this context, recent work suggests that YF-17D activates multiple TLRs (TLR2, TLR7, TLR8, and TLR9) on distinct subsets of DCs to stimulate a mixed Th1/Th2 profile (48). Distinct TLRs appear to differentially control the Th1/Th2 balance, thus, while MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D (48). In addition, YF-17D also activates RIG-I and MDA5 (Pulendran, unpublished

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observations). Furthermore, the attenuated Mycobacterium bovis strain BCG strain, which is used in the tuberculosis vaccine, and with which most infants in the developing world are vaccinated, has been shown to signal via TLR2, TLR4, TLR9, and DC-SIGN (49–51). Interestingly, while induction of TNF-a by DCs is dependent on TLR2 and TLR4, the cell wall component ManLam signals through DC-SIGN and inhibits TLR signaling in DCs. However, the consequences such signaling might have on adaptive immune responses induced by BCG need to be determined. Clearly, the elucidation of the innate immune receptors and cell types that mediate the efficacy of successful vaccines represents a critical challenge in development of future vaccines against global pandemics and emerging infections. Paradoxically, the vaccinia virus, which was administered to scores of millions of individuals during the smallpox eradication campaign in the 1960s and 1970s, infects DCs and inhibits DC activation, and causes extensive cell death, resulting in cross-presentation of cellular antigen (52). The mechanism of inhibition of DC activation is poorly understood, although two vaccinia virus open reading frames (ORFs) termed A46R and A52R, when expressed in mammalian cells, were shown to interfere specifically with IL-1 signal transduction. A46R partially inhibited IL-1-mediated activation of the transcription factor NF-kB, and A52R potently blocked IL-1-, TLR4-, and TLR3-mediated NF-kB activation (53). Licensed Adjuvants While live, attenuated vaccines contain microbial or viral stimuli that activate the innate immune system and in effect act as ‘‘their own adjuvants,’’ recombinant vaccines such as the Hepatitis B vaccine need to be administered with exogenous adjuvants. At present, alum and MF59 represent the only adjuvants licensed for clinical use in humans; however, the molecular basis of their adjuvanticity is not known (54). Alum consists of aluminum salts, which can be emulsified with the antigen to generate a gel-like substance. A widespread belief is that alum may exert a ‘‘depot effect,’’ whereby the emulsion retains antigen at the site of injection and releases it slowly to promote sustained antigen presentation (54). However, while this may offer a partial explanation of how alum functions, rigorous experimental evaluation of other possible mechanisms must be performed. For example, it has been shown that alum exerts a direct effect on IL-4-producing Gr1þ cells that were essential for priming and expansion of antibody-producing B cells in vivo (55). Furthermore, a recent study suggested that TLR signaling was not essential for the induction of antibody responses in mice immunized with alum plus antigen (56). However, two important points in this study should be stressed: the mice were deficient in one or more components of the TLR signaling pathway and thus may have developed compensatory mechanisms, and the antigen employed was actually a hapten conjugated to a large protein carrier, thus the findings might have limited applicability to a true vaccine response in a wild-type host. Interestingly, another recent study shows that alum induces caspase-1 [an essential component of the inflammasome (see above)] activation in human DCs and triggers the release of IL-1b and IL-18 (57). It is unclear precisely how alum results in caspase-1 activation. As discussed above, the NLR family consists of several proteins that could link pattern recognition to caspase-1 activation. Therefore, it is possible that one or more of the NLR family members mediate the effects of alum. MF59 was licensed for human use in Italy in 1997 and then in 20 other countries for use with the influenza vaccine in

the elderly (58). MF59 is essentially an oil-in-water emulsion, with the oil being squalene, a naturally occurring substance, which is an intermediate in the human steroid hormone biosynthetic pathway, and a precursor to cholesterol. MF59 probably works by facilitating uptake by professional antigenpresenting cells, including DCs, although whether there is also a stimulatory effect on the innate immune system remains to be determined. Monophosphoryl lipid A (MPLA) is an agonist of TLR4 that is being developed by GlaxoSmithKline (GSK) for use as a vaccine adjuvant. MPL is a derivative of lipid A from Salmonella minnesota and was recently licensed in Europe as a component of an improved vaccine for hepatitis B (Fendrix) (59). MPL is known to induce robust Th1 responses, with considerably reduced toxicity compared with LPS (59). In addition, MPL exerts potent adjuvant effects when administered via mucosal routes and when used in combination with other adjuvants such as alum (59). GSK Vaccines’ AS01A adjuvant consists of a TLR ligand in a saponin and liposome vehicle, AS02B is a TLR ligand in saponin emulsion, and AS04 is a TLR ligand in alum (59). Furthermore, a new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), has been engineered specifically to target human TLR4, and these mimetics are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. Emerging Adjuvants There is an intense effort to develop new adjuvants. Several companies are exploring the efficacy of using various TLR ligands in this regard. A few of these are briefly reviewed here. Others are discussed in more depth in chapter 25. TLR9 ligands. Coley and Dynavax are pursuing agonists of TLR9. Recently, CpG DNA has been administered with the hepatitis B vaccine Engerix-B to HIV-positive individuals who were previously nonresponsive to the Engerix-B vaccine. The addition of CpG induced rapid seroconversion and sustained maintenance of antibody titers (60). Moreover, in a recent phase Ib blinded, randomized, controlled clinical trial, coadministration of CpG ODN as adjuvant (CPG 7909, 1 mg) with a one-tenth dose of a commercial trivalent killed split influenza vaccine (Fluarix1) and resulted in similar levels of antigen-specific IFN-g secretion from restimulated peripheral blood mononuclear cells as were obtained with the full-dose vaccine administered without CpG ODN (58). In addition, Dynavax is pursuing a strategy to chemically link CpG DNA to the protein antigen with a view to targeting the antigen and adjuvant to the same antigen-presenting cell. Such a strategy results in a greatly enhanced immune response (61) and thus holds promise. Interestingly, even noncoding mammalian DNA complexed with cationic lipids is known to induce strong Th1 responses and antibody titers in mice (62). Whether this immunogenicity is due to TLR9 activation by CpG motifs in the DNA or whether additional mechanisms play a role needs to be determined. Toll-like receptor 7/8 ligands. Members of the imidazoquinoline family of ‘‘immune response modifiers’’ such as R837 and R848 have been shown to induce robust Th1 responses. 1 R837 (Imiquimod ) is used as a topical treatment for genital warts (63). Conjugation of TLR7/8 ligands to the recombinant protein antigen gag enhances the potency of the protein in inducing gag-specific CD8þ T-cell responses in nonhuman primates (64).

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Toll-like receptor 5 ligands. VaxInnate is exploring a strategy in which the gene encoding the TLR5 ligand flagellin is fused to genes encoding specific protein antigens. Immunization of mice with the recombinant proteins generated from such fusions resulted in potent antigen-specific B- and T-cell responses, including cross-priming of CD8þ effector T cells. The antibody response consisted of both the IgG1 and IgG2a isotypes, suggesting a mixed Th1/Th2 profile (65). Finally, the immune responses protected mice from lethal challenge with the relevant pathogen (influenza A virus, West Nile virus, and Listeria monocytogenes), protection that was not provided by immunization with the antigen alone or a simple cocktail of antigen and flagellin (65,66).

SUMMARY Recent advances in our understanding of the molecular basis of innate immune recognition represent a renaissance in vaccine discovery. The discovery of TLRs and appreciation of their signaling pathways and role in adaptive immune responses are the first step in this regard. Today, several TLR ligands are being developed as vaccine adjuvants. However, further understanding of the roles played by other innate immune receptors, including NLRs, CLRs, and RLRs, in innate recognition and translation to adaptive immune responses will undoubtedly provide new insights into the mechanism of action of our most successful vaccines and adjuvants. Furthermore, such understanding should permit increasingly rational choices about the best means of achieving the most optimal immunogenicity with the least toxicity.

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38. Wieland CW, Koppel EA, den Dunnen J, et al. Mice lacking SIGNR1 have stronger T helper 1 responses to Mycobacterium tuberculosis. Microbes Infect 2007; 9(2):134–141. 39. Dillon S, Agrawal S, Banerjee K, et al. Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. J Clin Invest 2006; 116(4): 916–928. 40. Chedid L, Lederer E. Past, present and future of the synthetic immunoadjuvant MDP and its analogs. Biochem Pharmacol 1978; 27(18):2183–2186. 41. Siddiqui WA, Taylor DW, Kan SC, et al. Vaccination of experimental monkeys against Plasmodium falciparum: a possible safe adjuvant. Science 1978; 201(4362):1237–1239. 42. Allison AC, Byars NE. Immunological adjuvants: desirable properties and side-effects. Mol Immunol 1991; 28(3):279–284. 43. Watanabe T, Kitani A, Murray PJ, et al. NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses. Nat Immunol 2004; 5(8):800–808. 44. Maeda S, Hsu LC, Liu H, et al. Nod2 mutation in Crohn’s disease potentiates NF-kappaB activity and IL-1beta processing. Science 2005; (307):734–738. 45. Henderson DA. The miracle of vaccination. Notes Rec R Soc Lond 1997; 51(2):235–245. 46. Monath TP. Yellow fever vaccine. Expert Rev Vaccines 2005; 4(4):553–574. 47. Napolitani G, Rinaldi A, Bertoni F, et al. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 2005; 6(8):769–776. 48. Querec T, Bennouna S, Alkan S, et al. Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity. J Exp Med 2006; 203(2): 413–424. 49. Tsuji S, Matsumoto M, Takeuchi O, et al. Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guerin: involvement of toll-like receptors. Infect Immun 2000; 68(12):6883–6890. 50. Geijtenbeek TB, van Vliet SJ, Koppel EA, et al. Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med 2003; 197(1):7–17. 51. von Meyenn F, Schefer M, Weighardt H, et al. Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guerin by Flt3-ligand generated dendritic cells. Immunobiology 2006; 211(6–8):557–565. 52. Norbury CC, Malide D, Gibbs JS, et al. Visualizing priming of virus-specific CD8þ T cells by infected dendritic cells in vivo. Nat Immunol 2002; 3(3):265–271.

53. Harte MT, Haga IR, Malony G, et al. The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense. J Exp Med 2003; 197(3):343–351. 54. O’Hagan DT, Valiante NM. Recent advances in the discovery and delivery of vaccine adjuvants. Nat Rev Drug Discov 2003; 2(9): 727–735. 55. Jordan MB, Mills DM, Kappler J, et al.. Promotion of B cell immune responses via an alum-induced myeloid cell population. Science 2004; 304(5678):1801–1810. 56. Gavin AL, Hoebe K, Duong B, et al. Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science 2006; 314(5807):1936–1938. 57. Li H, Nookala S, Re F. Aluminum hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release. J Immunol 2007; 178(8):5271–5276. 58. Podda A, Del Giudice G. MF59-adjuvanted vaccines: increased immunogenicity with an optimal safety profile. Expert Rev Vaccines 2003; 2(2):197–203. 59. Alderson MR, McGowan P, Baldridge JR, et al. TLR4 agonists as immunomdulatory agents. J Endotoxin Res 2006; 12(5):313–319. 60. McCluskie MJ, Krieg AM. Enhancement of infectious disease vaccines through TLR9-dependent recognition of CpG DNA. Curr Top Microbiol Immunol 2006; 311:155–178. 61. Kanzler H, Barrat FJ, Hessel EM, et al. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med 2007; 13(5):552–559. 62. Dow SW, Fradkin LG, Liggitt DH, et al. Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously. Immunol 1999; 163(3):1552–1561. 63. Ahonen CL, Doxsee CL, McGurran SM, et al. Combined TLR and CD40 triggering induces potent CD8þ T cell expansion with variable dependence on type I IFN. J Exp Med 2004; 199(6):775–784. 64. Wille-Reece U, Flynn BJ, Lore K, et al. HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8þ T cell responses in nonhuman primates. Proc Natl Acad Sci USA 2005; 102(42):15190–15194. 65. Huleatt JW, Jacobs AR, Tang J, et al. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 2007; 25: 763–775. 66. McDonald W, Huleatt JW, Foellmer HG, et al. Generation of an efficacious West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity. J Infect Dis 2007; 195:1607–1617. 67. Querec TD, Akondy RS, Lee EK, et al. Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol 2009; 10(1):116–125. Epub 2008 Nov 23.

19 Immunodominance, Deceptive Imprinting, and Immune Refocusing Technology Peter L. Nara Biological Mimetics, Inc., Frederick, Maryland, and Department of Biological Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa, U.S.A.

George Lin Biological Mimetics, Inc., Frederick, Maryland, U.S.A.

INTRODUCTION A striking disparity exists between the relatively small number of diseases currently prevented by available vaccines and the many infectious diseases for which no vaccine is available. Given the approximately 200 years of ‘‘modern’’ vaccine development, there are but 28 human vaccines licensed for use in the United States, and most of these are derived from very old classical technologies. But there are many infectious diseases for which traditional vaccine technologies fail to produce an effective vaccine. Interestingly, most of the viral and bacterial pathogens, for which successful human or animal vaccines exist (class I pathogens), share common genetic, transmission, and immunologic characteristics that make them good candidates for the early technologies of inactivation and attenuation developed many years ago. The same is not true of the class II pathogens. However, new technologies and conceptual breakthroughs may induce these pathogens to finally yield to vaccinologists. In this chapter, we focus on breakthroughs in understanding the nature of protective responses in the context of immunodominance.

THE LIVING HOST: A MICROBIAL GALAPAGOS ISLANDS Much like Darwin gleaned new biological and evolutionary principles from observing and studying evolutionary fitness, survival, and selection pressures on living things in specialized environments (the Galapagos Islands), a similar examination of how the vertebrate host immune system is sculpted from a complex interplay of self versus nonself, as well as the subsequent large degree of symbiotic and commensal microbial colonization may likewise yield new insights and principles applicable to class II pathogens. Immunology sprang from early studies in microbiology and pathobiology, and later itself spawned the discipline termed ‘‘vaccinology.’’ Early African, Middle Eastern, and Asian healers and medicine men around 1000 A.D., followed by a new generation that included Lady Mary Wortley Montague (1689– 1762), the Rev. Cotton Mather (1663–1728), Edward Jenner (1749–1823) among others, all observed the profound protection from infection, disease, and death of an animal or human if they were previously exposed to the powered, dried humors, or liquids of a microbe (1). Nobel prizes in immunology, from

the first in 1901 (von Berhing for work in diphtheria) to 1996 [Doherty and Zinkernagel for dual antigen recognition system of major histocompatibility complex (MHC) using lymphocytic choriomeningitis virus (LCMV) in mice], have their roots in experimental animal model systems. Experimentally introducing microbial pathogens into a host animal allows for the controlled induction and disturbance of the host’s cellular and humoral defense systems, which allows for further dissection and exploration of the effector functions responsible for clinical outcome. The early fundamental discoveries in vaccination and its use in health care worldwide stand as some of the most important medical and public health discoveries ever made. Important insights are gained by looking into the evolutionary selection pressures and host-microbial niches to which class I and class II pathogens have evolved (Figs. 1 and 2). In general, two distinct but overlapping physical, temporospatial, and host defense–related niches are observed for any given species, which pathogenic and nonpathogenic microbes might exploit. The first niche is the prenatal/early postnatal period, and given that the most basic tenet of life is reproduction, any given species in any given year will produce some number of offspring or face extinction. Thus, a supply of newborns and therefore immunologically naı¨ve offspring will be available for colonization and/or infection. Other than maternal antibody (colostrums, egg yolk, etc.), the evolutionary equivalent of maternal vaccination and passive transfer therapy of the newborn, the early postnatal period is a rather vulnerable period of time in the host, thus providing a well-defined cellular/humoral niche in which to evolve. As the maternal antibody wanes or an unfortunate offspring fails to receive adequate colostrum, the young host generally experiences increased susceptibility to infection, prior to the induction of any specific acquired immunity. Incumbent on the successful evolution of class I pathogens (in a relatively naı¨ve host immune system), would be to invest in a more stable genetics, with high replication/fitness, contagiousness, and efficient transmission profiles rather than investing largely on immune evasion strategies (2,3). The end game of class I pathogens appears to exist in the environment of available offspring by infecting young hosts efficiently, thus amplifying and spreading to new hosts in shorter time periods than class II pathogens. However, the period of time beyond the early postnatal period to the postreproductive period of the same animal

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Figure 1 Graphical representation of a typical course of infection of a host with a class I pathogen. Note that infection leads to initial period of pathogen replication/amplification, which reaches some critical threshold of immune activation, induction of protective effector activities (CTL, neutralizing antibody, etc.) leading to a fall in pathogen titer, elimination of the infectious pathogen, and the induction of long-lasting memory. Abbreviation: CTL, cytotoxic lymphocyte.

mentioned in the preceding text can be viewed as an extended opportunity for infection. The second niche, a temporally much longer one, can run all the way from the postnatal to the geriatric period. The host selection pressure(s) under these extended conditions are very different from those of the shorter early pre/postnatal period exploited by the class I pathogens. Unlike class I pathogens, class II pathogens are constantly changing in response to a host immune system, which is continuously being ‘‘educated.’’ Pathogens, depending on their infection and transmission dynamics (acute and reoccurring versus chronic), could now be faced with a greater pressure toward development of immune evasion strategies required over extended periods of time. Microbes capable of reinfecting and/or establishing long chronic active or chronic latent infections would now more likely be selected and thrive in this niche, thus emphasizing the greater return on any and all investments related to immune evasion. It is useful and interesting to think about ways in which the pathogen might accomplish this task: (i) it could simply evolve to be more selflike and therefore not easily recognized; (ii) it could evolve countermeasures to chemically and enzymatically inactivate the various effector activities the immune system mounts against it; (iii) it could invoke some form of immune suppression or modulation; and lastly (iv) it could try to outrun the immune system by evolving many antigenic forms. Most if not all of these survival strategies have been described in some form for some of these pathogens. There appears however, to be a fifth and possibly more cosmopolitan strategy, whereby some features of the above strategies are coupled to others in what has been termed ‘‘deceptive imprinting’’ (4) (see later in the chapter).

CLASSIFICATION OF PATHOGENS

Figure 2 Graphical representation of a typical course of infection of a host with a class II pathogen. Note that infection leads to initial period of pathogen replication/amplification, which sometimes is accompanied by a simultaneous induction of non-protective immune responses (cross-reactive recall, enhancing antibody, blocking antibody, cytokine axis deviation, etc.) and/or along with the induction of strain-restricted immune responses (CTL, neutralizing antibody, etc.). In the case of annual re-colonizers [depicted by the reoccurring peaks of pathogen titers (dotted gray lines)], this leads to a fall in pathogen titer and its temporary elimination, at least until the host is exposed to another antigenic variant of the infectious pathogen. In the case of chronic active/latent infections (solid black lines), the pathogen establishes a persistent infection with the host and eventually succumbs to systemic disease. Rare and low titers of broader neutralizing antibodies and CTLs are found in some hosts exhibiting these types of infections. Abbreviation: CTL, cytotoxic lymphocyte.

As mentioned earlier, the current stable of licensed vaccines and related technology in both the human and veterinary medical arenas are generally successful against the class I pathogens (3). Class I pathogens (such as measles, mumps, rubella, and distemper viruses ) (Table 1 and Fig. 1) are those pathogens that in general, (i) infect or cause the most serious disease in children/young adult individuals, (ii) carry a relatively stable microbial genome, (iii) have a natural history of disease, which results in spontaneous recovery, and (iv) induce durable memory, associated with polyclonal and multiepitope antigen recognition. In contrast, class II pathogens (Table 2 and Fig. 2), such as influenza virus, human immunodeficiency virus (HIV) type 1, malaria, tuberculosis, trypanosomiasis, schistosomiasis, leishmaniasis, Anaplasma sp., enterovirus, astrovirus, human rhinovirus, Table 1 Characteristics of Class I Pathogen and Successful Vaccines l

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Pathogen usually infects narrower host age range (e.g., newborn or pediatric) Natural history of disease exhibits spontaneous recovery and long-lasting immunity Production of neutralizing antibody and cytotoxic lymphocyte Priming with wild type whole killed or attenuated pathogen induces protective immunity Vaccine does not dysregulate immune response—induces solid memory/anamnestic response Pathogen shows limited antigenic variation Immune response is directed to multiple epitopes

Chapter 19: Immunodominance, Deceptive Imprinting, and Immune Refocusing Table 2 Characteristics of Class II Pathogens l

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Pathogens infect wider age ranges of a given host (e.g., young, adolescent, adult, and geriatric) Annual recolonizers (infect multiple times per year) Chronic active infections Chronic latent/reactivation Natural history of disease fails to induce long-lasting immunity No memory, poor memory Short-term type specific memory Immune suppression Disease enhancement Pathogen shows high degree of focused regions of antigenic variation, immunodominance, and convergence of B- and T-cell epitopes Early dominant nonprotective/strain restricted immune responses Immune dysregulation Evidence for preexisting immunologic reactivity (B- and T-cell level) Early cross reactive recall A more limited oligoclonal response directed at fewer immunodominant epitopes

respiratory syncytial virus, foot and mouth disease virus (FMDV), norwalk viruses, toxigenic/pathogenic E. coli, Neisseria sp., Streptococcus sp., nontypeable Haemophlius influenzae, hepatitis C, cancer cells, etc., are characterized by quite opposite features (3). For example, they (i) tend to infect and be transmitted in a significantly extended host age range, with infections occurring and reoccurring from childhood through the geriatric period; (ii) exhibit microbial genetic instability in defined regions of their genome (a hallmark of their successful evolution); (iii) in some cases, include spontaneous recovery of disease that frequently still leaves the host vulnerable to multiple reinfections and/or the establishment of either a chronic-active or chronic-latent state; (iv) induce oligoclonal early immune responses that are directed to a very limited set of immunodominant epitopes; and (v) cause immune dysregulation following infection or vaccination. This encompasses a growing set of immunologic mechanisms associated with the phenomena of ‘‘deceptive imprinting’’ (Table 3, to be discussed in Immunodominance section). These mechanisms include (i) the formation of blocking antibodies— these antibodies produced earlier and directed to noncritical epitopes (decotopes) that interfere with the subsequent recognition of antibodies to other epitopes (4) (Fig. 1), (ii) MHC-blocking

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antibody, which are directed to T-helper (TH) and cytotoxic T lymphocyte (CTL) epitopes, interfering with their proteolytic processing and MHC presentation (5–8), (iii) atypical primary immune response-Ig subclasses, (iv) epitope-mediated T regulatory (reg)/suppressor activity, (v) anamnestic cross-reactive recall, and (vi) skewing of the TH1 or TH2 cytokine axis. These mechanisms, together and separately, appear to be largely responsible for the limited success in developing broadly effective and safe licensed vaccines for class II pathogens. This appears to be due, in large part, to our naı¨ve understanding of the fundamental laws governing the development of the vertebrate host defense system, its origins, repertoire development, maintenance, activation, and senescence. Also lacking is the very important effect on the hosts’ innate and adaptive immune systems due to the coevolution of the host-associated microbiological environments to be discussed later. Any such models of immunology must take explicit account of the dynamics of the primary repertoire of the 108 distinct B-cell and T-cell sequences that exist within each individual with a theoretical capacity of responding to up to 1.6
1013 eliminons (8). Such large numbers suggest that should pathogens decide to outrun the immune system, a large degree of continuous genetic variation would be needed, thus in many cases costing the pathogen in various fitness gains/losses, possibly resulting in the pathogen expending much of its potential antigenic and functional capital in a few host generations. Thus, a different immune-evasion strategy would need to be evolved to conserve some core of genetic stability, while at the same time distracting the immune effector activity of the host just enough to permit multiplication.

IMMUNODOMINANCE The terms ‘‘immunodominance’’ and ‘‘immunodominant’’ were originally used to describe prominent humoral responses to various antigens in the late 1960s and early 1970s (9–11). Immunodominance was then described soon after the discovery of the MHC and restriction (12) but a decade prior to the discovery that antigenic peptides are presented by MHC molecules (13). A working definition of immunodominance would be the focusing of humoral and cellular immunity toward one or just a few antigenic determinants even during immune responses to complex microorganisms, cells, or antigens (Figs. 3 and 4). Immunodominance as a major mechanism of immune evasion was not recognized until somewhat recently (15). Although described in many experimental immunology systems, the controlling mechanisms of determinant immunodominance are only just beginning to be appreciated, especially in relation to the interplay between infectious organisms, the acquired immune system and vaccine development (2–4,14,15).

Table 3 Characteristics of Deceptive Imprinting Immune dysregulating responses Predominant TH1 or TH2 cytokine, cellular profiles Atypical Ig class induction (primary IgG vs. IgM) Steric blocking inhibitory antibody Enhancing antibody Inhibitory T helper antigen processing antibody HLA-DM editing of CD4 cells and B cells Loss of antigenic epitope specificity Cross-reactivity (B- and T-cell level) Promiscuous T-cell reactivity Original antigenic sin-like recall (B and T cells) Heterologous recall

Immunodominance of T Cell Determinants It was originally noticed that the CTL responses to antigens such as the minor histocompatibility antigen (MiHA) H-Y and LCMV were restricted by a single or just a few H-2 haplotypes (12,13). More detailed studies of CTL responses to mouse sarcoma virus (MSV) (16), influenza A viruses (IAVs), (17,18) and Epstein-Barr virus (19–21) revealed that responses were strongly linked to mouse H-2 alleles. Using recombinant inbred mouse strains expressing different H-2 genes and then transplanting lymphomas as targets, Gomard et al. (16) identified H-2Kd as the major restriction allele for anti-MSV CTL responses, whereas the H-2Dd allele was not involved in these responses.

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Figure 3 The major stages and factors involved in sculpting the host adaptive immune repertoire. (A) The four major stages whereby the host B- and T-cell repertoire of the acquired immune system is sculpted. (B) The resulting major clonotypes resulting from the previous sculpting.

Doherty et al. (18), using similar mouse strains and fibroblast cell lines as CTL targets, defined H-2Kb as a ‘‘nonresponder’’ MHC gene locus in anti-IAV responses despite this being a responder in anti-recombinant vaccinia virus (rVV) responses. These observations were made as part of the demonstration of MHC-restriction of CTL responses and constituted the first indication of H-2 allele– associated immunodominance. Immunodominance has also been found to regulate CTL responses to bacteria (22) and tumor antigens (23–25). On the basis of the detailed studies of in vivo and in vitro CTL responses toward MiHAs, it appears that only a very small proportion of epitopes, probably less than 10%, is dominant (20,26–29). A priori, immunodominance may not appear advantageous, as it signifies that by focusing exclusively on one or a few epitopes, the immune system places all its eggs in the same (or a few) basket(s). Theoretically, this should increase the risk that pathogens or tumor cells can escape from immunosurveillance. Therefore, it is of great importance to decipher the rules that govern immunodominance to understand why the repertoire of T-cell responses is restricted to only a few determinants when confronted with numerous immunogenic epitopes, and what are the implications of this restriction. Similarly, vaccinologists are interested in knowing whether presentation of dominant epitopes is equivalent to presenting an entire antigen, and the relative advantages and disadvantages of this vaccine approach. It is now well established that antigen-specific immunodominant responses are linked to particular MHC alleles (30–33), known initially as ‘‘immune response (Ir)’’ genes (34,35). From

such beginnings, it was soon found that even within the strains that responded to a given virus, the CTL responses were highly focused on just a few proteins and often on a single polypeptide (36). These ‘‘immunodominant responses’’ were traced to a subregion of the protein antigen (37) around the time when the peptide nature of antigenic determinants was being elucidated (13,17,38). Nearly simultaneously, Sercarz and colleagues (39) studying both B-cell and CD4þ T-cell responses to an artificial antigen, hen egg lysozyme (HEL), observed that the CD4þ T-cell responses to some determinants were easily detectable, whereas responses to other determinants were much smaller and consequently harder to demonstrate. There were yet other determinants that were not detected under normal circumstances unless very high levels of antigen were used for priming. Determinants involved in detectable responses were defined as either immunodominant determinants (IDDs) or subdominant determinants (SDDs) depending on their reproducibility and magnitude; the third category of undetectable determinants were called cryptic determinants (39), a term derived from their earlier studies on anti-HEL B-cell responses and autoimmune responses (40– 42). Similar phenomena were observed in mouse models of IAV, LCMV, herpes simplex virus (HSV), and Listeria monocytogenes (LM) infection (Table 4). In a given mouse strain, the major responses to these pathogens were often directed toward a single IDD. However, progress in unraveling the factors that controlled immunodominance was limited by the lack of tools for immune monitoring. The field accelerated

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Figure 4 Recognition of pathogen-derived antigens by the adaptive immunity. (Dark gray box) Parts of the immune system influenced by immunodominance. The black arrows indicate possible other pathways that immunodominance may operate in the host defense system. For explanation see text. Abbreviations: APC, antigen-presenting cell; BCR, B-cell receptor; MHC, major histocompatibility complex; TCR, T-cell receptor. Source: From Ref. 14.

when novel methods for enumerating TCD8þ arrived in the late 1990s. These technologies included MHC-peptide tetramers (43) and intracellular cytokine staining (ICS) of antigenspecific T cells (44). These new techniques allowed accurate enumeration of specific T cells in combination with their surface and functional markers in the absence of in vitro T-cell expansion (45,46). Moreover, the newer methods were up to 100-fold more sensitive in detecting Ag-specific TCD8þ

than the established limiting dilution analysis that measured CTL precursor frequencies indirectly through target killing (47,48). This observation suggested that historical estimates of Ag-specific TCD8þ numbers might have been drastically underestimated. Since then, many experiments have reassessed these systems and revised the estimates of Ag-specific TCD8þ numbers (46,49). The newer technologies were not only more sensitive in detecting the immunodominant

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Table 4 Nature of Immunodominance l l l

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For some antigens, the host immune system exhibits an ‘‘aleatory nature’’ (predisposed nature/propensity) Antigenic hierarchy is a fact of immunologic life Some multideterminant antigens exhibit high degrees of epitope immunodominance and restriction at both the B- and T-cell level across genetic backgrounds Certain amino acid sequences as well as primary, secondary, tertiary, and quaternary protein structures are more immunogenic than others Immunodominance of epitopes can greatly influence the response to other subdominant epitopes Multiple levels of B- and T-cell repertoire sculpting occurs in a given host that can be exploited by a pathogens Germ line-self/nonself—reactive clones to self Postnatal bacterial colonization—mucosal and integumentary Environmental—food, water, air, soil, etc. Infections with other viral, bacterial, parasitic, fungal pathogens

TCD8þ but, particularly in the subdominant TCD8þ, were more readily appreciated. It was then possible to quantitate TCD8þ responses reproducibly and define ‘‘immunodominance hierarchies’’ (50,51). Another form of T-cell skewing and immunodominance is described for the non-classical MHC heterodimer called HLA-DM (52). MHC class II molecules on the surface of specialized APC present peptides to CD4 T lymphocytes, and thus select a useful CD4þ T-cell repertoire in the thymus and guide CD4þ T-cell responses to pathogens in the periphery. DM, a nonclassical MHC heterodimer, functions as a chaperone to promote capture of diverse peptides by MHC class II molecules inside the endocytic compartments of APC. In addition to releasing CLIP (class II–associated invariant chain peptide) from newly synthesized MHC class II and stabilizing empty MHC class II (52), DM edits the repertoire of non-CLIP peptides loaded onto the MHC class II binding groove (53,54). CLIP is the part of the invariant chain that binds MHC II groove and remains there until the MHC receptor is fully assembled. The purpose of CLIP is to prevent the binding of an antigen prematurely, which would disrupt the synthesis of MHC II. Peptide editing by DM is thus consequential for recognition of exogenous and endogenous Ags because it introduces prejudice toward display of DM-resistant and/or DM-dependent peptides by MHC class II molecules. Features of the peptide-MHC complex that determine whether DM would release the peptide from the MHC II-peptide complex or enhance its binding remain highly controversial. Although initial studies suggested that DM susceptibility of a peptide is a correlate of low intrinsic stability of its binding to MHC class II (54), many recent studies have concluded to the contrary that the intrinsic kinetic instability of a peptide-MHC complex does not predict DM susceptibility of the peptide and vice versa (54). Results of a recent study show that DM is required for skewing the CD4 T-cell immune response toward immunodominant epitopes in vivo. Although multiple elements, such as differential peptide affinity for MHC and differential enzymatic processing, within APC could be responsible for anointing a single peptide in a 312 amino acid a protein kinase C-like molecule on the surface of the Leishmania parasite (54), DM peptide editing was a major determining factor leading to the expression of the immunodominant phenotype in both the BALB/c and B10. BR strains of mice. DM-deficient mice were unable to elicit T cells reactive with either the immunodominant LACK 158 to

173 in H-2d mice or the DM peptide editing on CD4 immunodominant LACK 81 to 96 in H-2k mice, indicating that these epitopes were DM dependent (rather than simply DM resistant) for their display by MHC class II molecules. Thus, HLADM peptide editing, during presentation of a pathogen or a protein Ag in vivo, can be envisioned to translate into a prejudiced display of some peptides (DM dependent/resistant) and an exclusion from presentation of other peptides (DM susceptible) by antigen-presenting cells (APCs). The peptide-editing properties of DM could thus sculpt the outcome of an in vivo T-cell immune response in two ways: (i) by expression of a biased spectrum of peptides by thymic APC and a consequent maturation of an altered T-cell repertoire in the host (53,55), and (ii) by display of selected peptides by macrophages and dendritic cells (DCs) recruited in the host defense against pathogens and proteins. Tetramers, or multimers of MHC/peptide complexes, allowed assessment of specific T cells at various stages of their development and differentiation following antigen-specific activation; another key technological advance was the development of T-cell receptor (TCR) transgenic (Tg) mice expressing a ‘‘monoclonal’’ T-cell repertoire (56). Transfer experiments involving TCR Tg-T cells enabled these T cells to be tracked directly ex vivo at early stages of the immune response, and also permitted the in vivo study of T cell-T cell (T-T) and/or T cell– antigen-presenting cell (T-APC) interactions. A large body of work has since shown how immunodominance might be controlled or influenced by the steps involved in the creation of antigenic peptides (Ag-processing) and their presentation by the MHC-I class molecules addressing the potential contribution of antigen processing and presentation to the establishment of immunodominance hierarchies (44–56). Thus, when confronted with numerous peptides presented in the context of self-MHC, T cells usually respond only to a limited number of peptide epitopes. Context is the general term used to describe the influence of various factors within the intact antigen or pathogen that have been associated with controlling immunodominance. These are independent of the primary sequence of the peptide under study, and include protease susceptibility or resistance of the antigen, typically within the residues that flank the peptide, a feature that may potentially control the release from the intact antigen during endosomal processing. Additionally, context refers to the additional peptides within the antigen that compete for binding to MHC class II molecules,

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tertiary structure of the antigen, and copy number of the peptide or antigen. In many studies of immunodominance, the activity of the endopeptidases has been suggested to modulate determinate display on the surface of APCs. Early studies showed that treatment of the APCs with inhibitors of endosomal proteolysis could either enhance or inhibit antigen presentation, depending on the epitope under analysis, arguing that resident endosomal proteases can alternatively promote or diminish peptide availability. Immunodominant epitopes tend to cluster in limited regions of the antigen under study, often residing in exposed or protease-sensitive loops. These types of data suggest that antigens have a high degree of secondary and tertiary structure, while other studies have demonstrated that CD4þ T-cell epitopes are preferentially associated with structural stable regions and overlapping B cell epitopes (38). Either way, the immunodominance effect considerably restricts the repertoire of T-cell effectors. Overall, it appears that numerous pathways are involved in the generation of immunodominance (DM peptide-editing function, etc.), which sculpts the specificity and immunodominance in the CD4þ T-cell response to an Ag. These may have evolved to conservatively focus the T-cell response to a selected few epitopes of a pathogen. Such a strategy could be protective for the host on one hand because it prevents cross-reactive autoimmune responses (57), but detrimental on the other because it allows a pathogen to exploit antigenic variation and immune escape (58). We speculate that the evolutionary pressures to focus T-cell responses to fewer epitopes may have outweighed the disadvantage to the host due to potential immune escape by the class I pathogens during an immune response directed to one or only a few epitopes; however, class II pathogens appear to have evolved and usurped various aspects of the antigen processing cellular pathways leading to overt immunodominance, which may interfere with the host’s ability to eliminate or prevent reinfection with these pathogens. These insights could reveal opportunities for developing new approaches and tools to diversify the epitope specificity of a T-cell response when an in vivo T-cell response targeted to multiple specificities is beneficial to the host.

Immunodominance of B-Cell Determinants The immunodominance of B-cell epitopes, although recognized historically earlier than the T-cell phenomena, has not been as extensively advanced and researched as T-cell dominance. Unlike T cells, B cells recognize many different determinants on the surface of a various foreign and self antigens directly, thus presenting a technically challenging problem to vaccinologists interested in mapping and studying the interaction between antibody and epitopes on complex glycoprotein/protein shapes. The chemical aspects of antigen– antibody binding have been thoroughly reviewed and discussed by Getzoff et al. (61). Most determinants depend on the conformational integrity of the native protein (60) and those to which an individual responds is dictated by a complex set of rules, which have not yet been fully elucidated (61). Some molecular characteristics of immunogenic and dominate epitopes have however been elucidated and include (62) (i) surface accessibility of binding sites, (ii) hydrophilicity (which obviously overlaps with (i), (iii) flexibility, since the more flexible protein segments are able to bind by induced fit in an antibody combining site that is not perfectly complementary to the

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native structure, (iv) S-S loops and kinks in the protein, (v) presence of certain preferred amino acids, (vi) propensity for variation (substitution, deletion, etc.), (vii) disorder, (viii) the presence and location of N-linked carbohydrates, and (ix) proximity to sites recognized by TH cells. TH epitopes of HIV, for example, are clustered within hot spots of the HIV glycoprotein gp120, and many of these T-cell epitopes overlap with known antibody-binding domains (63). Similarly, studies of influenza and parainfluenza membrane proteins have shown that immunodominant TH-cell epitopes are localized to exposed protein surfaces (63–65). So far, the significance of the proximity including the overlapping of TH-cell and B-cell epitopes and antibody-binding sites is not clear (62). Theoretically, an almost infinite spectrum of antibodies can be induced by a pathogen, yet they are not, especially with class I pathogens. However, similar to the T-cell response, only a small number of the many specifically reactive B-cell clones in a given individual are recognized, favoring the selection of dominant B-cell epitopes. Antigenicity of individual determinants on a polypeptide Ag is generally thought to constitute a surface static property of the Ag (66,67). Consequently, attempts at ab initio identification of B-cell epitopes, as mentioned in the preceding text, have primarily focused on structural aspects of polypeptide Ags. These analyses rely on assessment of individual domains either for their surface accessibility, flexibility, or structural propensity (62,67–69). However, although both repertoire plasticity and a requirement for epitope accessibility are required, earlier results indicate that the spectrum of epitopes recognized in an Ag-specific humoral response constitutes only a fraction of that which may be predicted using current methods (61–73). These studies have demonstrated that, subsequent to the initial recognition of Ag, immunological events operate to restrict the range of epitope-specific responses that are retained (74,75). Of the diverse clonotypes activated upon first exposure to Ag, these selection processes ensure that only those B cells with highest affinity for Ag are selected for seeding of germinal centers (GCs) and consequent retention in the response. Thus, the pre-GC phase selection appears to constitute the first and, perhaps critical, filtering step in the pathway. Although surface accessibility is undoubtedly a prerequisite, recent results (75–79) now suggest that downstream, immune-mediated mechanisms will also need to be taken into consideration to explain the hierarchical immunodominance of B-cell epitopes on protein antigens (72,80–83). An intriguing observation pertaining to this issue was described for the immunodominance of the amino acid sequence diphenylaminofluorene (DPAF) in segment PS1 of the Hepatitis B antigen when placed in the context of a variety of alternate sequences (76). It was found to be independent of the position of the PS1 sequence in the Ag, the nature of the flanking domains, and also the genetic background of the mouse strain employed, thereby implying quantitative differences in B-cell recognition, the DPAF segment was always the most immunodominant among the various epitopes presented by the Ag (76). The fact that in this case the antibody population engendered by an antigen to a given immunodominant epitope, such as DPAF is independent of the overall location, strongly implies immunogenicity, and the parameters involved in defining immunogenicity of a multi-determinant Ag are independent of those mechanisms that influence inter-epitopic hierarchy on the same Ag. A single residue glycine substitutions within the

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DPAF epitope was found to result in a redistribution of epitope specificities in favor of alternate determinants within the PS1 segment. Although the avidity of the resulting responses was markedly reduced, this finding was consistent with those suggesting that the immune system can be refocused, a topic that will be discussed later in the chapter in more detail. These latter results provided a possible clue by implicating that the affinity/avidity of a primary response to a multi-determinant Ag may be epitope dependent.

WHERE DOES THE IMMUNODOMINANCE COME FROM? As has been discussed in the sections above, immunodominance at both the B- and T-cell levels appears to involve many segmental pathways involved in many, if not all, antigen presentation pathways. In addition to there being multiple steps in the antigen pathway during which immunodominance may be exploited by a pathogen, it also appears that the B and T cells themselves, as they are derived from the preimmune to the immune repertoire are also, in a way possibly ‘‘set up’’ to contribute to the ‘‘aleatory quality’’ (derived from the Latin word ‘‘alea’’—randomness) of immunodominance in the immune system (Fig. 3).

Prenatal Germ Line Period We now know that Burnet’s paradigm (reviewed in Burnet, F. M. (1959) The Clonal Selection Theory of Acquired Immunity, Cambridge University Press, Cambridge, U.K.), centered on the elimination of auto-reactive lymphocytes, is only partly correct since T cells specific for self antigens are part of the healthy immune repertoire. Immunodominance in some cases appears to be developed for some epitopes at the onset of immunological ontogeny in the host during repertoire sculpting events of the prenatal period when self versus nonself is being sorted out and cross reactive self clones are established either through mimicry or direct shape complementarities. Antibody repertoire development during fetal life has unique characteristics compared with postnatal life, because it is a period when exposure to external antigen has not yet occurred and immune recognition is mainly directed to autologous antigens (84). Three distinct characteristics of the fetal repertoire have been described, namely restriction in antigen specificities, low avidity, and multireactivity to self-antigens. Previous reports have indicated that restrictions in antigen specificities are achieved by limited and preferential usage of certain VH and Vk gene segments (85,86). Preferential gene use reflected the proximity of gene segments in chromosomal position (85,86). Fetal diversity was also reported to reflect limited diversification of the CDR3 region during early ontogeny. This was related to a relative paucity of N region additions (87,88), a high frequency of homology-directed recombination, and generation of shorter CDR3 domains during the fetal life (89). It has been suggested that the restricted diversity of the fetal Ig repertoire predisposes to the generation of multi-reactive, low affinity self-reactive antibodies at this stage of development (90). The physiologic impact of self-reactive, germ line– encoded Abs remains obscure, but it has been suggested that self-reactivity of these antibodies is an essential component for B-cell survival and maturation (91), and mediates positive selection (92). In addition, cross-reactivity with exogenous

microbes may play a role in host defense to the small number of pathogens that may threaten the survival of an individual during the perinatal period (93–95). In accord with the above concepts, in vivo data studying myelin basic protein, so-called MBP-reactive T-cell clones shows that T cells specific for immunodominant determinants— even on self antigens—may in some cases have more likely been expanded by nonself-like epitopes, and will recognize as optimal ligands peptide sequences derived from microbial proteomes. This system would in some cases ensure a broader protection profile against pathogens, with reduced probability of cross-activating potentially autoreactive T cells. A danger signal may be further reduced by the possibility that T cells with high-affinity receptors for self epitopes be deleted in the thymus, leaving a peripheral repertoire of potentially autoreactive T cells that respond to self peptides in the low-affinity range. Qualitative self-nonself discrimination at the TCR level may therefore be unnecessary, since the compositional bias toward the nonself proteome in immunodominant T-cell epitopes optimizes the immune response against pathogens and the maintenance of tolerance. The lack of major qualitative differences between human and microbial proteomes implies repeated encounters with cross-reactive epitopes. Hindrance for self-nonself discrimination may instead help the maintenance of tolerance and sustain the mature repertoire of naive (96) and memory (97) subsets. Moreover, the comparability of the similarity profile of amino acid sequence between self and nonself may help to explain the paradox of thymic positive selection that occurs on a self ‘‘substrate,’’ but must serve in the response toward nonself (97–104). The fact that self-like epitopes tend to be suboptimal ligands (as was shown with the MBP-specific T-cell clones) confirms that mimicry can be functional both to the maintenance of memory and to positive selection. Nonetheless, a T lymphocyte can no longer be regarded as ‘‘autoreactive’’ or ‘‘pathogen-reactive’’ on the basis of the stimulating antigen alone. The probability of mimicry at the epitope level should be taken into account when studying the defensive or auto-reactive potential of a T lymphocyte for vaccine design or tolerance induction. In the preimmune repertoire, T-cell precursor frequencies for a given antigen have been estimated to be on the order of 1:1,000,000 or less (104), but such estimates are very crude and limited to a few antigens derived form MHC-tetramer assays. Despite the paucity of direct data on precursor frequency in the preimmune repertoire, it is likely that processing and class II-restricted presentation of self-antigens or those closely related to host homologues can dramatically skew the developing T-cell repertoire that is available to respond to antigenic challenge (105–107). It is now clear that many selfantigens gain access to the thymus and therefore influence T-cell repertoire selection.

Postnatal Bacterial Colonization Period Right after birth, under normal circumstances, the postnatal period is characterized by a massive colonization of the integument and oral gastrointestinal tract, primarily with various types and classes of bacteria. Thus, the next level in which the immune repertoire and immunodominance may be established is during this postnatal period in which large amounts of foreign bacterial protein and carbohydrate antigens are presented. It is well established that the bacteria that colonize the

Chapter 19: Immunodominance, Deceptive Imprinting, and Immune Refocusing

intestine after birth are important stimuli for the developing immune system. The establishment of the intestinal microflora commences immediately after birth and proceeds in a sequential manner until a complete microflora, consisting of more than 400 bacterial species, is obtained at two to three years. A number of factors may influence this process, including delivery and feeding mode, social contacts, and the degree of environmental hygiene (108). For instance, there is a rapid increase in serum immunoglobulins and secretory IgA production during the first weeks or months after birth (109,110). Studies in animals show that continuous acquisition of new bacterial strains in the microflora is required to keep the immune system in an activated state (111,112). Furthermore, the presence of an intestinal microflora has been shown to be essential for the efficient induction of oral tolerance in animals (113). Experimental studies have suggested that microbes and their products may increase the suppressive potential of CD25þ Tregs (113). Commensal bacteria have been shown to influence gene expression and immune function in the intestine. Using DNA microarrays, Hooper et al. (114) found that Bacteroides thetaiotaomicron, a prominent component of the normal mouse and human intestinal commensal microflora, can modulate the expression of enterocyte genes involved in several important intestinal functions, including nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation when germ-free mice are colonized. Umesaki and Setoyama (115) summarized the immunological effects of indigenous flora, including induction of MHC class II and fucosyl glycoconjugates in intestinal epithelial cells (IECs); expansion of alpha-beta TCR –bearing intestinal epithelial lymphocytes and acquisition of its cytotoxic activity; development of Peyer’s patches; expansion of IgA-producing plasmas cells in the lamina propria; enhancement of the function of macrophages and neutrophils; and the induction and maintenance of oral humoral and cellular immune tolerance. Recently, Macpherson and Uhr (116) explained how DCs selectively induce IgA. They found that commensal-loaded DCs, which can retain small numbers of live commensal bacteria for several days, are restricted to the mucosal immune compartment by the mesenteric lymph nodes. This localization ensures induction of an IgA response without affecting systemic immunity. This period is characterized by a wide array of exposure to bacterial polysaccharides, lipoproteins, proteins and bacterial DNAs having toll-like receptor agonist activity (117). It is interesting to note that children exhibit poor reactivity to bacterial polysaccharide vaccines. Conjugating these polysaccharides to proteins greatly enhances their immunogenicity and protective immune responses. Leishmania Model In early work using the Leishmania susceptible/resistant mouse models to dissect the TH-1 and TH-2 immune axis, a remarkable set of data was accrued. Following a parasitic infection in a susceptible mouse strain, a rapid (8–16 hours) clonal expansion of a memory CD4 TCR Va8 and Vb4 helper T-cell immunophenotype positive responses was observed, responsible for driving a dominant TH-2, interleukin (IL)-4 driven axis (118). This resulted in hypergammaglobulinemia and a highly focused and nonprotective IL-4-driven B-cell response, leading to rapid and fatal parasitemia. All this immunologic and immunodominant activity was found to be directed and mapped to a

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30 amino acid epitope located in a protein kinase C-like protein on the surface of the infecting parasite, termed the LACK antigen (118). Subsequent studies identified the primary, secondary and immune memory events that were directed to a bacterial antigen epitope found on the surfaces of commensal bacteria colonizing the intestines of BALB/c mice (119). The most interesting finding (in this model), however, was observed when the LACK antigen epitope was functionally immune dampened by making a LACK epitope Tg BALB/c mouse that was tolerant to the 30 amino acid immunodominant epitope, LACK. This resulted in a major shift from a TH-2 to a TH-1, gamma-interferon, IL-2-driven response and complete protection from parasitic challenge (118). This finding suggests that the sculpting of the host repertoire, as discussed above can have a profound effect on a subsequent exposure to another pathogen.

Postnatal Antigen Exposure Period (Environmental) Cross-reactivity Between Different Virus-Specific T Cells, Alloantigens, and Allergens The degenerate nature of antigen recognition by the TCR is exemplified by the ability of T cells to recognize nonself or allogeneic MHC molecules. Allospecific T cells represent a substantial population of the naı¨ve T-cell repertoire, with between 0.1% and 10% of naı¨ve T cells within an individual host being reactive with any unique allogeneic haplotype, as measured by limiting dilution analysis and quantitative measurements of cells responding to alloantigens in vivo (120–122). This high frequency of allospecific T cells allows a host to efficiently generate effector allospecific T cells following exposure to alloantigens in the form of blood transfusions, prior transplants, or pregnancy (123). Surprisingly, memory allospecific T cells are detectable in patients that have never been obviously exposed to alloantigens (124,125), suggesting that these T cells were activated by cross-reactive environmental antigens. Early studies in murine models demonstrated that CD8 T cells activated during an acute LCMV infection of H2b mice recognized both H2k and H2d allogeneic target cells in a standard cytotoxicity assay (126). Cytotoxic, allospecific CD8 T cells were also generated following infection with Pichinde virus (PV), vaccinia virus (VV), and mCMV (126). Allospecific CTL activity was also detected in humans infected with Epstein-Barr virus (EBV) during acute infectious mononucleosis (127–130). These results showed that allospecific CD8 T-cell responses are activated after viral infections, but did not address whether the allospecific responses were activated by an antigen-dependent cross-reactive mechanism or by a nonspecific bystander mechanism mediated by the massive production of cytokines after infection. We now know that many virus-specific CD8 T cells generated in response to viral infections directly cross-react with alloantigens. Short-term CD8 T-cell clones derived from LCMV-infected mice were shown to lyse both virus-infected syngeneic targets and uninfected allogeneic target cells. This cross-reactivity between LCMV-specific T cells and alloantigens is consistent with earlier studies in which long-term murine CD8 T-cell clones specific for either influenza virus or vesicular stomatitis virus (VSV), and human clones specific for EBV recognized allogeneic cells in cytotoxicity assays. In addition, alloreactive T cells specific for H2Kk could be shown to lyse syngeneic H2b-target cells infected with influenza virus (125).

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Cross-reactivity between virus-specific CD8 T cells and alloantigens has been visualized directly from mice acutely infected with LCMV (126). LCMV-specific CD8 T cells isolated from H2b mice produced IFN-g following a short in vitro stimulation with either H2d- or H2k-expressing cell lines. This cross-reactivity was broad-based, as a portion of T cells specific for each of the four LCMV-epitopes examined (GP33, NP205, GP276, and NP396) cross-reacted with H2d, yet it was distinctive, as different proportions of each epitope-specific population recognized H2d or H2k targets. Together, these findings demonstrate the promiscuity of alloantigen recognition in a variety of viral systems. Heterologous immunity is the term used to describe the phenomenon by which memory T cells that were generated during an earlier infection are reactivated in response to a second, unrelated infection. Typically, the onset of hepatitis C virus (HCV) infection is asymptomatic, and persistent infection develops despite the presence of a CD8 T-cell response. In a study by Urbani et al., two patients with a very rare fulminant onset of HCV infection displayed an unusual CD8 T-cell response that was unprecedented in its strength and narrow focus (131). About 36% and 12% of all peripheral blood CD8 T cells from these respective patients targeted a single epitope within the HCV nonstructural protein 3 (NS3) and also crossreactively recognized an IAV neuraminidase epitope with close sequence similarity (131,132). In contrast, patients with nonfulminant onset of HCV infection displayed a broader, multispecific CD8 T-cell response of lower magnitude (133,134). The authors concluded that exposure to IAV, as confirmed by cellular immune responses against a second influenza A virus epitope, preconditioned the CD8 T-cell response to HCV and focused it on a single cross-reactive epitope. The result was severe immunopathology. The notion of cross-reactivity was supported by the demonstration that only those T cells that bound HCV NS3 epitope–MHC tetramers produced interferon-g and increased cell surface expression of the degranulation marker CD107a in response to stimulation with the crossreactive IAV epitope (135–137). In contrast, no response was observed upon stimulation with an unrelated, non-cross-reactive IAV epitope. The cross-reactive nature of the response was further confirmed by the demonstration that those T cells that did not bind HCV NS3 epitope–MHC tetramers did not respond to stimulation with the cross-reactive IAV epitope (134). As immunodominance may be a relative and context dependent event in the immune system, suppressor activity of other epitopes relative to those epitopes that are not being suppressed will be found to be immunodominant. Regarding environmental antigens, the collective results indicate that the control of TH2 immune response against naturally exposed harmless environmental antigens is mediated by Tr1 cells in humans. Effector (allergen-specific TH2) and suppressor (allergen-specific Tr1) T cells exist in both healthy and allergic individuals in certain amounts. Their ratio determines the development of a healthy or an allergic immune response (138,139). These data may explain the spontaneous development and spontaneous healing of allergic diseases. Although in low frequency, the existence of potential suppressive allergenspecific Tr1 cells in allergic individuals suggests a possible way of treatment. The knowledge of this cellular and molecular basis is pivotal in understanding the mechanisms of immune tolerance or allergy development against harmless environmental proteins. There is clear evidence from various animal models and human studies for an active mechanism of immune suppression,

whereby a distinct subset of T cells inhibits the activation of conventional T cells in the periphery (140–142). This Tr cell population has been determined to be CD4þCD25þ T cells. They can prevent the development of autoimmunity, indicating that the normal immune system contains a population of professional regulatory T cells. Elimination of CD4þCD25þ T cells leads to spontaneous development of various autoimmune diseases, such as gastritis or thyroiditis, in genetically susceptible hosts. The frequency of CD4þCD25þ Tr cells is ~ 10– 15% of CD4þ T cells, whereas the frequency of IL-10–secreting T cells of single allergen specificity ranges between 0.1 and 0.007% of CD4þ T cells. This shows that the frequency of single allergen-specific Tr1 cells, which are also CD4þ CD25þ, ranges between 1 in 1,000 and 1 in 20,000 of the whole CD4þ CD25þ Tr cell population. Although many aspects of the mechanisms by which suppressor cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of IL-10 and transforming growth factor b (TGF)-b (143). Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4þ T cells in the presence of IL-10 in vitro (144). The nonspecific T-cell suppressor activity of IL-10 and TGF-b has been consistently reported in experiments with high amounts of exogenously added suppressor cytokines (144–146). However, the present work demonstrates that Tr1 cells display antigen-specific suppressor activity in very low numbers. If the number of cells exceeds a threshold that provides sufficient quantities of suppressor signals, apparently they show nonspecific suppression. Depending on their frequency, the first T cell that contacts the APC may be very critical in the subsequent decision to stimulate or suppress the specificimmune response. If the first T cell to contact the APC is a Tr1 cell, it may silence or regulate the maturation of APC. IL-10 downregulates the antigen-presenting capacity, such as HLA-DR expression, costimulatory molecules, and several cytokines in DCs and monocytes/macrophages (145,146). Recently, differentiation of a distinct dendritic cell subset in the presence of IL-10 has been demonstrated that induces tolerance through the generation of Tr1 cells (144,146). In addition, exposure of mature pulmonary DCs to respiratory allergens stimulated the development of Tr1-like cells, which was dependent on dendritic cell IL-10 production (147). Together, these findings suggest that IL-10-secreting T cells may regulate the functional state of APCs in a way that these APCs can now promote the generation of Tr1 cells.

THE THEORY OF DECEPTIVE IMPRINTING Deceptive Imprinting Now that a general outline has been established of what immunodominance is at both the B- and T-cell levels, it is useful to now put this in the context and interface of the immune system and microbial pathogens, as discussed earlier in the section with class II pathogens where they appear to have evolved a combination of mechanisms for immune evasion in which immunodominance is coupled to immune regulation and antigenic variation. Immunodominance has been found to regulate CTL, CD4þ helper T cells and B-cell responses to viruses, bacteria, parasites, tumor antigens, and MiHAs. Based on detailed studies of in vivo and in vitro CTL responses toward MiHAs, it appears that only a very small proportion of epitopes, probably less than 10%, are dominant. A priori, immunodominance may not appear advantageous, as it

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signifies that by focusing exclusively on one or a few epitopes, the immune system places all its eggs in the same (or a few) basket(s). Theoretically, this should increase the risk that pathogens or tumor cells can escape from immunosurveillance. Therefore, it is of great importance to decipher the rules that govern immunodominance to understand why the repertoire of T-cell and B-cell responses are restricted to only a few determinants when confronted with numerous potential or proven to be immunogenic epitopes and what the implications of this restriction are. These fundamental differences in the pathogen classes appear to equate to very different outcomes in vaccine induced protection when using conventional and current vaccine technologies. The major differences appear mainly due to the evolution and presence of immunodominant non-protective epitopes (IDNPEs) on class II pathogens, which appear to misdirect and prevent the primary immune response to be initiated simultaneously against multiple epitopes on the pathogen (1–3). An example of an IDNPE (the GH-loop of an ungulate class II pathogen RNA virus) as it exists on a complete viral structure is shown in Figure 4. Note the proximity of the decoy to the conserved integrin and sulfated glycosaminoglycan cellular receptor binding ligands. This pattern of very close adjacent location of IDNPE to highly conserved receptor binding ligands is observed in a number of other viral bacterial and parasitic examples and suggests a conservation of structural relationship. The reason for this structural relationship between the IDNPE and the conserved ligands of the pathogen may lie in the evolution of the antibody binding paratope and the presence of blocking antibody. Blocking antibody is defined as a binding antibody produced by the host in response to an infection with a class II pathogen directed to an IDNPE that interferes with the generation of other antibodies that would otherwise be directed at conserved and functional determinants needed by the pathogen for infection and or replication or transmission. It seems that the molecular distances observed in our studies very closely approximates that of the footprint of an epitope and paratope interaction. Thus locating IDNPE close to more conserved functional critical determinants needed by the pathogen may induce an effective form of steric hindrance. These IDNPEs can be of B and/or T cell in origin and appear to act as immunologic decoys and are dispensable for replication during the evolution of the virus in the host. In essence this means that the same, or a highly related set of immune evasion strategies will be (as described earlier) adhered to whether the pathogen is a more genetically simpler RNA virus, like that of human rhinovirus, influenza viruses, HIV-1, hepatitis C, FMDV of ungulates or more genetically complex pathogens like those of malaria, tuberculosis, or cancer. An analogous morphological survival adaptation is operational in nature in many predatorprey relationships involving protective coloration. Deceptive coloration more specifically ‘‘Aggressive mimicry’’ and ‘‘false eyespot,’’ or ocellus is used for example (analogous to IDNPEs). The Original Theory and discovery of the phenomena of deceptive imprinting began in the late 1980s to early 1990s while working on HIV-1 and has some of its roots in the older theory of ‘‘original antigenic sin,’’ first described in 1960 by Thomas Francis, Jr. in the article On the Doctrine of Original Antigenic Sin (148), regarding observations made in swine influenza. Recently this concept has helped to formulate the foundations for developing new approaches and technology for overcoming the phenomena as well as theory and insights into how sequence space localization in the immune system,

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response to vaccination and disease helps to predict epitope analysis in highly mutable pathogens, to efficacy for vaccine design in influenza and many other pathogens, as well as how glassy dynamics in the adaptive immune response prevents autoimmune disease (149). In this view, the combined use of genomic instability and antigenic variation associated with a pathogen’s evolution are coupled to selective immunodominance of B-and T-cell epitopes, antigenic hierarchy, laws of mass action, and immunoregulatory feedback circuits as an immune-evading strategy. All of this appears to be coordinated and designed to misdirect the induction and control the innate as well as the effector and memory phases of acquired immunity against the pathogen. Deceptive imprinting is the evolving evolutionary principle and immunologic phenomena that states that a class of successful pathogens (viral, rickettsial, bacterial, fungal, protozoal, parasitic, cancer cells) have evolved IDNPEs, so-called decotopes to recall, induce, misdirect, misuse, and block the innate and adaptive immune systems leading to ineffectual, nonprotective, immunoregulatory, strain-restricted protection, and/or disease promoting/enhancing activity at the B- and T-cell level. This keeps the host from fully recognizing and directing its attack against the more conserved functional elements of the pathogen, such as those necessary for its initial adsorption, binding, penetration and/or release, colonization of and/or transmission. This results in a loss of immunologic specificity, reduces the polyclonal and affinity maturing complexity of the immune system resulting in some cases in a form of immunologic amnesia.

IMMUNE REFOCUSING TECHNOLOGY Many antigens considered for use in vaccines will contain several epitopes, some of which may be advantageous for the production of neutralizing antibodies while others may stimulate responses that are harmful to the host or that are unable to elicit the desired protection. For example, the unwanted epitopes may cross-react with self-proteins in the host, leading to an autoimmune response as in the case of Trypanosoma cruzi and nervous tissue (150). They may undergo rapid mutation and thus fail to elicit a broadly protective immune response [e.g., HIV gp120, malaria, African trypanosomiasis, or suppress an otherwise protective immune response (e.g., Mycobacterium leprae and M. tuberculosis)] (149). In such situations, it would be highly desirable to exclude the unwanted parts of the antigen. In the case of T-cell epitopes this may be achieved by the construction of multiple antigenic peptides containing only the beneficial T-cell epitopes in a tandem array to form a synthetic polypeptide (for a recent review see Ref. 151). As discussed above, for B-cell epitopes it is more difficult because the epitopes are usually discontinuous, and therefore require correct folding of the antigen to form the immunogenic region of the protein. Though synthetic peptides containing part of the epitope have been used, the high entropy of such peptides in solution coupled to the relatively small percentage of the molecular structure represented by a peptide from a whole protein antigen produces antibodies with relatively low affinity for the native antigen (152). An alternative strategy is to develop epitope-specific molecules by selectively mutating the targeted immunodominant epitope without affecting the overall folding of the polypeptide chain. This was first accomplished for a complex glycoprotein by Nara and colleagues at the National Cancer

Immunogenicity

Immunogenicity

P5 and P2 fimbrae

P5 VP2

gp135 envelope

gp120/41

gp120 V3()

Nontypeable Haemophilus influenzae

IPNV

CAEV

HIV-1

Immunogenicity

Preclinical challenge

Immunogenicity

Immunogenicity Studies mice, pigs

Broader neutralization Infected cell/heterologous protection Virus challenge—mouse model Enhanced neutralization good CD8 responses Decreased hypergammaglobulinemia

Moderate heterologous neutralization

Some heterologous protection Good heterologous neutralization Heterologous challenge-good protection from disease Very good heterologous neutralization Good heterologous protection from disease

In vitro neutralization Challenge pending APHIS, Plum Island Modest improvement in heterologous epithelial binding assay

Protection from parasitic challenge/ disease Challenge studies in chickens

Result

Nature Immunol 2004; 5:934–942

IP developed J Immunol 1997; 159:279 Publication in preparation J Immunol 1998; 160:5676 J Virol 2002; 76:4222–4232

IP being developed J Virol 2004; 78:9190 Publication in preparation

Publication in preparation J Immunol 2005; 171:1978–1983 IP being developed IP being developed

IP being developed

IP being developed Pending publication IP being developed

Science 1996; 274:421

IP/publication

Abbreviations: FMDV, foot and mouth disease Virus; IPNV, infectious pancreatic necrosis virus; CAEV, caprine arthritis encephalitis virus; HIV, human immunodeficiency virus; LCMV, lymphocytic choriomeningitis virus; LACK, Leishmania analogue of the receptors of activated C kinase. Source: Ref. 156.

GP 61-80

Chinchilla challenge studies Immunogenicity Field trial studies

GH-loop VP1 capsid

FMDV

LCMV

First generation immunogenicity studies

24K antigen

Poultry Coccidia

Murine immunogenicity challenge studies Immunogenicity

LACK ag

Leishmania

Status

Target

Pathogen

Table 5 Current Summary of Immune Refocusing Proof-of-Concept Studies

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Figure 5 Priming and selective clonal expansion of CD8 T-cell specificities. After infection, the cell presents antigenic peptides on the cell surface in association with MHC class I molecules. CD8 T cells with specific receptors become activated. Immunodominant epitopes induce a strong T-cell response whereas subdominant epitopes induce weak responses. The histogram illustrates an antiviral CD8 T-cell specificity (immunome) analysis after murine cytomegalovirus (mCMV) infection. Source: From Ref. 14.

Institute while trying to develop an effective HIV-1 vaccine (153) and has been applied effectively for a number of other experimental antigens including the development of an antifertility vaccine for humans in which extensive sequence homology with LH results in the production of LH crossreactive antibodies when hCG is used as an immunogen (154) (Table 5) (reviewed in Refs. 2 and 3). Using a platform technology called ‘‘immune refocusing’’ (Fig. 5), which seeks to map, identify and then remove or immunologically mask unwanted IDNPEs via site-directed mutagenesis strategies. Once immune-dampened, the immune system can now be

allowed to resurvey the remaining antigenic hierarchy of a given antigen possibly leading to enhanced immunogenicity of the other determinants on the protein antigen and thereby refocusing and enhancing the response to subdominant or cryptic epitopes. By design these second and third tier epitopes are naturally less, or in the wild-type state not immunodominant molecular structures. Once the immune system is unbiased during the immunogenic priming event from IDNPE, new or enhanced immune responses are usually measured to previously lower titered and/or new epitopes, with the hope that these second and third tiered epitopes are

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Figure 6 Fully, molecularly solved structures of the HA of human influenza and entire virus of foot and mouth virus (FMDV) showing the relative locations of the IDNPEs (decotopes) and cellular binding ligands. (Panel A) The monomeric ribbon structure and locations of the IDNPEs of the HA globular head. The gray ribbon (See area ‘‘D’’) is another IDNPE and not the portion of the sialic acid binding site as depicted in panel B. (Panel B) The oligomer (trimetric space filling model) and the relationship between two of the IDNPEs and the conserved sialic acid cellular binding site (Panel C) The location and the proximity of only one (GH-loop) of the four IDNPEs located on the viral capsid and the conserved cellular binding regions. The molecular separation is less than 15 A˚. Source: From Ref. 155.

now part of the critical molecular (structural infrastructure) (e.g., proteins/glycoproteins used in adsorption, binding entry and or release for cells) and may be more protective against heterologous challenge. The left side of the Figure 4A shows strainspecific antibodies (in red) binding to IDNPEs. Once the epitopes are identified and mapped, they can be modified by various means both intramolecularly (e.g., deletion, amino acid substitution, insertion of self motifs or introduced N-linked sequons resulting in post-translation N-linked glycosylation) (Figure 4B). If the modifications are done subtly, the structure of the molecule

can be preserved to retain biological activities (such as cell fusion and receptor/coreceptor binding), and therefore conformation of the critical epitopes. The new antigen now induces antibody responses to previously less immunogenic epitopes, and thus appears to offer a technology capable of inducing broadened immunity against heterologous pathogens. As noted in Figure 4, the novel refocused antigens can be used to screen more efficiently combinatorial libraries and/or induce and select for therapeutic and diagnostically more useful monoclonal antibodies (Figs. 6 and 7). (Reviewed in Ref. 156).

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Figure 7 Immune refocusing technology. A representative antigen for immune focusing is depicted as the influenza hemagglutination protein. (Panel A) IDNPEs are depicted in light gray and type-restrictive antibodies in dark gray. Immune refocusing is shown in this example by the introduction of an N-linked sequon leading to the specific addition post-translationally of a complex carbohydrate to the protein chain in the IDNPEs, resulting in masking of the IDNPE to the host immune system and an antigen that now is capable of inducing newer antibodies throughout its structure. (Panel B) An actual glycoprotein that has been immune dampened by the introduction of two N-linked glycosylation sites (lane M). Note the shift in size of the glycoprotein compared to the wild type unmodified glycoprotein (lane W). To confirm that glycosylation has occurred, the panel on the right demonstrates the shift toward a smaller protein size following the treatment with glycosidase A deletion of the IDNPE is shown in lane T as indicated by the smaller size of the glycoprotein. From Ref. 156.

SUMMARY Deceptive imprinting and immune refocusing technology could represent a major paradigm shift, opening up new areas and understanding in innate host defenses, immunology, microbiology, structural biology, as well as vaccine, diagnostic, and therapeutic development. The dynamic interplay of host and pathogen in the class II pathogen game appears to have evolved a new and exciting set of principles and rules that future immunologists, vaccinologists, molecular biologists, and microbiologists will need to better understand in their quest for developing novel prophylactic and therapeutic immunobiologics. If accomplished this could provide for novel broadly protective immune compositions, therapies and/or novel and complimentary strategies to inducing enhanced resistance to multitudes of pathogens. New IDNPE databases and experimental/structural data sets will need to be collected and constructed from animal and human infections. Current data analysis so far argues strongly that pathogens exhibiting antigenic variation, genomic instability and escaping host immune defenses, are doing so primarily at the level of primary immune induction and regulation of effector, memory as well as suppressor/reg function and not simply by genetically ‘‘out-running’’ the immune system through antigenic variation. To date, some common immunophysical chemical themes of these IDNPEs are beginning to emerge along with the observation of their frequent positioning adjacent to and usually at certain fixed molecular distances to very conserve and critical cellular

ligand-binding domains necessary for the pathogen survival. Additionally, preliminary work suggests that some if not many of the origins of this immunodominance may lie in crossreactive (mimicry) of pre-existing immune responses derived in the host’s naturally sculpted B and T-cell repertoires. These repertoires are now known to be actively influenced from the earliest stages of development, such as cellular differentiation signals (the maintenance of self from nonself and neoplasia, etc.) during prenatal development to postnatal events such as bacterial colonization of the so-called normal flora throughout the body, exogenous foods, and environmental antigens and other acquired infections. This insight, if correct, would suggest that a given host develops a form of housekeeping B- and T-cell repertoires in its immunome, which leaves it protected from some pathogens while uniquely susceptible to others. This theory and technology presents a completely new and different insight into how we might look at and manipulate the host’s immune system—quite opposite to that of the last 200 years, as done by conventional vaccination. For example, it might be that the most robust protection is induced by (i) preventing or blocking a given exposure to a specific foreign environmental antigen or defined epitope; (ii) inducing a state of epitopespecific tolerance/suppression to the IDNPE, or (iii) presenting a well-defined IDNPE during early pre- or postnatal development leading to clonal deletion of specific decoy recognition, and thus the host’s immune system might profoundly alter its susceptibility to a given pathogen. It may also lead us to

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consider now treating a chronically infected patient (hepatitis C, malaria, HIV-1) with an idiotypic therapeutic antibody directed specifically at the proper clonally expanded IDNPE of B- and/or T-cell clones, thus deleting them from the active repertoire, while at the same time immunizing with a properly refocused antigen, which jump-starts and resets the immune system toward now recognizing and eliminating the offending infectious or cellular pathogen. Economically important food producing animals may be given IDNPEs early during their development and/or made Tg to the decoy epitopes, thus inducing a state of tolerance and avoiding their immune system from succumbing to the deceptive imprinting response.

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20 Standardization and High-Throughput Measurement of T-Cell Responses to Vaccines Kent J. Weinhold and Janet S. Ottinger Departments of Surgery and Immunology and Duke Translational Research Institute Immune Monitoring Core, Duke University Medical Center, Durham, North Carolina, U.S.A.

INTRODUCTION Most present day phase I/II clinical vaccine trials aim to develop more effective preventive or therapeutic vaccine strategies for infectious diseases and cancer. These studies have at their foundation a comprehensively designed immunologic assay plan that will measure both quantitative and qualitative aspects of the vaccine-induced immune responses. At the same time, they must permit reliable comparisons to the immunogenicities of vaccine strategies that preceded them. During the past 15 years, the field of vaccine research has witnessed an informational and technological explosion that has served to refine vaccine strategies and develop highly complex tools for assessing vaccine immunogenicity and a link to efficacy. Many of these advances have been driven by the critical need to develop an effective AIDS vaccine that could impact on the worldwide epidemic. Although the immunologic ‘‘correlates of protection’’ against HIV infection have yet to be elucidated, most in the scientific community feel that a vaccine is needed that will elicit long-lived and broadly reactive humoral and cellular immune responses, both systemically and mucosally. For the currently licensed human vaccines in use today, the role of vaccine-induced T-cell responses in preventing infections or disease has been less well established than the role of neutralizing antibodies, although in many instances the basis for protection by these vaccines has never been clearly delineated.

CHALLENGES ASSOCIATED WITH MEASURING VACCINE-INDUCED T-CELL RESPONSES Since most developmental vaccine strategies are built upon the concept of making incremental improvements on existing vaccines, the assays used in assessing immunogenicity must provide reliable comparative data. This aspect of vaccine testing demands that highly standardized and formally validated assays be used in providing immunologic end points. The assessment of vaccine-induced T-cell responses in the setting of phase I/II clinical trials presents formidable challenges that do not apply to routine serologic immunogenicity testing, although standardization of the latter is of equal importance. All too often, optimization and standardization efforts are focused on the sophisticated technologies used to measure antigen-specific T-cell responses, with very little attention paid to an important aspect of immunogenicity testing, the overall functional integrity of the clinical specimens, in most

cases peripheral blood mononuclear cells (PBMC). No matter how potentially powerful or comprehensive the tools used in assessing the cellular immune responses, if the clinical specimen itself has been compromised by improper collection, transport, preparation, and storage, the eventual immunologic measurement, even those made using a highly standardized and validated assays, can be meaningless and misleading, since it represents at best an underestimate of true immune reactivity. Just as the assay platforms themselves must be validated using standard operating procedures (SOPs) that cover every detail associated with the technology, so too must the specimen acquisition, preparation, and storage procedures be standardized and formally validated. Considerations in developing a specific assay plan for phase I/II testing of T-cell responses elicited by a candidate vaccine should include the anticoagulant used in collecting the specimen, testing fresh versus cryopreserved specimens, real-time versus batch testing of specimens, specimen storage and transport, and, finally, the immune assay that can best address the relevant aspects of the vaccine-induced responses. Because of the complicated logistical considerations in getting clinical specimens to the testing laboratory in a manner that best preserves their functional integrity, especially in the context of multicenter vaccine trials, most present day vaccine networks have come to rely on testing of cryopreserved PBMC in a batch test mode using multiple timepoints for individual participants. While SOPs for viable cryopreservation and, perhaps more importantly, specimen thawing are essential, data published from the HIV Vaccine Trials Network (HVTN) Central Immunology Laboratories clearly identified one of the most critical elements in preserving the overall functional integrity of PBMC specimens is the time delay between specimen collection and PBMC isolation and cryopreservation (1). According to this report, overnight shipment to a centralized specimen-processing laboratory resulted in significant loss of antigen-specific functional reactivity compared with specimens that were processed and cryopreserved within eight hours of blood draw. Thus, for multicenter vaccine studies, standardized/validated specimen preparation, cryopreservation, and liquid nitrogen storage at the individual clinical sites represent the preferred method of handling. Specimens can subsequently be shipped to the testing laboratory using a liquid nitrogen dry shipper according to a validated SOP. As noted above, the process of thawing cryopreserved specimens prior to

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immunologic testing can be even more important than cryopreservation itself, and this procedure must be performed in a consistent manner according to validated SOPs. Since cryopreservation, no matter how meticulously performed, places stress on the population of immunocytes contained within PBMC, most immunogenicity assays presently employ an overnight culture of thawed specimens to permit stressed cells to undergo apoptosis, thus yielding a more accurate enumeration of viable cells that will be analyzed in the context of the specific immunoassay of choice.

STANDARDIZATION AND VALIDATION OF T-CELL ASSESSMENT OF VACCINE RESPONSES The application of these powerful new assay technologies to the specific evaluation of vaccine-induced T-cell responses carries the requirement for greater standardization and formal validation. Although not yet formally mandated by regulatory agencies such as the FDA for phase I/II clinical vaccine trials, certain network sponsors, including the Division of AIDS (DAIDS) within the NIH National Institute of Allergy and Infectious Diseases, have required that immune monitoring be conducted in strict compliance with Good Clinical Laboratory Practice (GCLP) guidelines (2,3) and according to validated SOPs. Formal assay validation procedures are based on documentation of multiple assay parameters, such as accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), resolution, specificity, linearity and range, ruggedness/robustness, and system suitability. The compilation of datasets that addresses each of these parameters becomes the validation package for each individual end point assay or procedure. Of equal importance to the quality control procedures required of each assay is the need for continuous quality assurance evaluation at regular intervals through external proficiency-testing programs such as those established by the Cancer Vaccine Consortium (CVC) and DAIDS. Adherence to assay standardization/validation is an essential component in testing the immunogenicity of different vaccine strategies, since it provides greater confidence that differences in measured immunologic end points are meaningful. Since many different networks and even more individual testing laboratories are involved in the phase I/II evaluation of novel vaccine strategies for cancer and infectious diseases, the importance of assay standardization and, more globally, assay ‘‘harmonization’’ across the many laboratories internationally cannot be overemphasized. As noted recently by Britten and coworkers (4), in the field of cancer immunotherapy there have, to date, been no consistently identified immune responses that track with clinical outcomes, perhaps due in part to the high variability in assay results. If cellular immune responses are to ever achieve the status of a ‘biomarker’ capable of predicting clinical benefit, better efforts must be made to ensure the highest achievable level of standardization, validation, and harmonization in cellular immune assays across all testing laboratories. At least two nonprofit organizations, the CVC of the Sabin Institute and the Cancer Immunotherapy (CIMT) Monitoring Panel of the Association of Immunotherapy of Cancer in Germany, are actively involved in instituting rigorous international efforts to harmonize cellular assays through extensive proficiency-testing programs. The combined efforts of these two networks have recently been published in an effort to better establish more stringent harmonization guidelines for T-cell-based immune monitoring assays (5,6)

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SELECTION OF ASSAY PLATFORMS FOR HIGH-THROUGHPUT MEASUREMENT OF VACCINE-INDUCED T-CELL RESPONSES Throughout the 1980s and 1990s, the assay technologies for measuring cellular immune responses began to migrate away from the once mainstay ‘‘bulk’’ assays such as 51chromium release (as a measure of lytic activity) and 3H-thymidine incorporation (as a reflection of antigen-driven proliferation) to what are now referred to as ‘‘single-cell’’ assays in which the responses of individual cells could be detected using various imaging techniques such as flow cytometry. The single-cell assay technologies available today for quantitative and qualitative measurement of vaccine-induced T-cell responses in phase I/II clinical trials are translational in nature, most having their developmental origins in basic research laboratories performing rigorous immunologic measurements in the context of animal model systems. For example, in 1983 Czerkinsky and coworkers developed a solid-phase enzyme-linked immunospot (ELISPOT) assay for determining the frequency of antibody-secreting cells (7). This technology was later adapted for measurement of IFN-g production by murine T cells (8) and, in 1997, eventually configured to enumerate antigen-specific human T cells (9). Since that time, the IFN-g ELISPOT assay has become a mainstay for quantitating antigen-specific T-cell responses to a wide variety of candidate immunogens (10–14). Perhaps the greatest technical advance of the past 10 years has been the development of both the equipment and reagents necessary to perform complex polychromatic flow cytometric analyses of highly defined cell subpopulations (reviewed in Ref. 15). As the field of clinical flow cytometry evolved beyond single- and two-color analyses in support of relatively simple, yet highly standardized CD4 and CD8 determinations, the field of immunology began assimilating new information regarding lymphocyte maturation and function, resulting in the development of new conceptual definitions of naı¨ve (N), central memory (CM), effector memory (EM), and terminal effector (TE) subpopulations within both CD4 and CD8 lymphocyte subsets (16,17). As novel flow cytometric technologies produced important new analytic tools and strategies, it ultimately became possible to combine assessments of cellular phenotypes and function (e.g., intracellular cytokine or chemokine production, degranulation), resulting in the current state-of-the-art strategy termed ‘‘polychromatic flow cytometry’’ (PFC), aimed at delineating multiple antigen-specific functions within highly defined lymphocyte subpopulations (18). An additional flow cytometry–based strategy for enumeration of antigen-specific CD8 cells was developed in the mid1990s by Altman and colleagues (19). In this assay, the frequency of CD8 cells capable of recognizing specific peptide-major histocompatibility complex (MHC) class I allelic tetramer complexes can be accurately quantified in mixed cell samples such as PBMC. While this highly standardized and amply validated assay strategy has great utility in following the actual levels of peptide-specific CD8 cells following vaccine administration, it depends on a priori knowledge of both the specific epitope as well as the MHC class I restricting allele, the latter requiring HLA typing of vaccine recipients. Therefore, this strategy has limited utility in the routine immunologic monitoring of vaccine-induced responses unless the vaccine strategy itself involves known ‘‘immunodominant’’ epitopes restricted by commonly expressed MHC class I alleles. Lastly, while tetramer or related assays measure the actual binding of epitope/ MHC complexes to the T-cell receptor, this process does not

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Table 1 Attributes of Single-Cell T-Cell Assay Platforms Available for Clinical Vaccine Studies Assay

IFN-g ELISPOT assay

Multimer binding assay

Cytokine flow cytometry

Sensitivity Advantages

1:10,000–1:50,000 Highly reproducible, most amenable to high-throughput batch testing analyses, measures T-cell function

1:10,000 Highly standardized and reproducible, can be used to capture viable, antigen-specific cells for further analytic measurements

Disadvantages

Normally formatted to only detect production of single cytokines (usually IFN-g)

Special considerations

Requires automated plate reader and associated software

Adaptability for high throughput

Medium

Does not measure lymphocyte function, requires a priori knowledge of epitope- and MHC-restricting alleles Requires flow cytometer and possibly cell sorter (for cell isolations) High

1:10,000 Capacity to measure multiple T-cell functions simultaneously within highly defined lymphocyte subpopulations Not yet fully adapted for highthroughput analyses, highly complex and time-consuming data analysis requirements Requires flow cytometer with polychromatic capabilities Medium

Abbreviations: IFN, Interferon; MHC, major histocompatibility complex.

provide insight into the functional reactivity of the epitopespecific cells. Although there are a number of potential single-cell assay platforms available for monitoring T-cell responses in the context of current clinical vaccine trials, not all are suited for highly standardized, high-throughput approaches. The relevant attributes of the three assay platforms most amenable to current immune monitoring strategies are presented (Table 1). Noticeably absent for this list is the carboxyfluorescein succinimidyl ester (CFSE) assay for measuring antigen-specific T-cell proliferation at the single-cell level (20). Although this assay platform has shown great utility in various basic and clinical research settings, it has not yet been adequately standardized and validated for entry into the realm of phase I/II clinical vaccine monitoring. Harmonization efforts for the CFSE assay are presently underway within the CVC.

ELISPOT Assay Platform The IFN-g ELISPOT assay was the first formally validated platform to be used in quantitating T-cell responses as the primary immunologic end point in a phase II study of a candidate AIDS vaccine (10). From many different aspects, the ELISPOT platform appears ideally suited for monitoring vaccine-induced T-cell responses (Table 1). The validated assay is highly reproducible, can be used with cryopreserved cells, and has been estimated to have the capacity to detect as low as one IFN-g-producing cell in 50,000 PBMC, a level of assay sensitivity required in evaluating new vaccine strategies that may have low T-cell immunogenicity. Since the assay is formatted to detect antigen-specific memory T-cell responses, peptide stimulation is usually performed throughout an overnight or 18-hour incubation period. The assay format will accommodate stimulation by single peptides as well as peptide pools on the basis of the components of the vaccine immunogen. A particularly useful variation on the normal IFN-g ELISPOT procedure is the use of peptide pools (usually 15- to 18-mer peptides with 11 amino acid overlaps) in a ‘‘matrix’’ arrangement that permits identification of specific peptides across a relatively large vaccine antigen such as HIV Gag (21). Important parameters that require attention as part of the validation process include intra-assay variability among replicates, inter-assay variability, performance variability among the technical staff, and inter-laboratory

variability when multiple testing laboratories are participating in a clinical vaccine trial. Many of these items can be addressed by inclusion of an assay control consisting of cryopreserved PBMC from a donor with known reactivity against a particular recall antigen such as cytomegalovirus (CMV) pp65. By obtaining and cryopreserving a viable leukapheresis product from such a donor, several hundred individual vials can be stored and subsequently used for each assay run. In this manner, a trend line of reactivity can be established in which the results on these samples assayed over time can be plotted and analyzed for unacceptable variances that may reflect subtle alterations in one or more assay components or procedures. In addition to a routine negative (i.e., no antigen) control and a mitogen-driven positive control that should reflect the gross functional integrity of the test sample, each assay should also include a positive control reflective of antigen-driven reactivity within the test PBMC. Routine controls of this nature could include CMV pp65 or, in the case of CD8 responses, a pool of epitopic 9-mers representing immunodominant epitopes in CMV, Epstein-Barr virus, and influenza viruses restricted by commonly expressed MHC class I alleles. The ELISPOT assay is a purely quantitative platform that measures the overall frequency of IFN-g within either a mixed population of PBMC or purified population of CD4 or CD8 lymphocyte subpopulations. It is important to note here that there remains substantial uncertainty as to whether qualitative, quantitative, or combined qualitative/ quantitative measures of T-cell responsiveness best translate into actual vaccine efficacy. By far the greatest limitation of the ELISPOT platform is its capacity to measure single-cytokine (optimized for IFN-g) production. Although there have been efforts to reformat the assay platform to simultaneously measure the production of multiple cytokines, these highly exploratory efforts have not generally undergone formal validation and subsequent implementation for following vaccine-induced T-cell responses.

Cytokine Flow Cytometry Platform The PFC platform emanates from extensive technologic advances in instrumentation and reagent development over the past 10 to 15 years. In its simplest form, PFC represents a highly quantitative tool much like the ELISPOT platform in which the frequency of cells responding to an antigenic stimulus can be

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Figure 1 Gating scheme for 11-color ICS/maturational panel. The first three gates are designed to identify singlets and viable CD3þ T-cells. Singlet gating is used to reduce the likelihood that false-positives might be generated as a result of coincidence for any two or more cells passing in front of the laser simultaneously. A multiplexed exclusion channel, comprising anti-CD14 þ anti-CD19 þ Violet Fixable Live/Dead amine binding dye, is plotted against CD3 to identify viable CD3þ T cells. From the CD3 gate, there are 2 basic subset anchor gates (CD4þ and CD8þ), followed by 10 maturational gates, five for each of the 2 basic subsets: naı¨ve (N), central memory (CM), effector memory (EM), effector (E), and terminal effector (TE). For each of the 10 maturational gates, there are 4 functional gates (IFN-g, TNF-a, CD107, and IL-2), resulting in a total of 40 functional gates. Each single set of 4 functional gates combine to generate 16 boolean gates. Each of the 16 boolean combinations consist of 5 polyfunctional subsets (1 subset positive for all 4 functions simultaneously, and 5 subsets positive for any 3 of the 4 functions but negative for the remaining one function); 6 bifunctional subsets (positive for any 2 of the 4 functions and negative for the remaining 2 functions), 4 monofunctional subsets (positive for only 1 of the 4 functions and negative for the remaining 3 functions), and one nonfunctional subset (simultaneously negative for all 4 functions).

readily obtained as a measure of cell function. The next level of analytic capability is captured in the definition of the responding T-cell population according to the broad categories of CD4þ or CD8 þ subpopulations. With the development of PFC reagents and instrumentation, the phenotypic definition of the responding T-cell populations can be further delineated into N, CM, EM, E, and TE populations through the use of sequential gating strategies (Fig. 1). Importantly, this strategy also permits qualitative assessment of antigen-driven responses reflected by the number of cellular functions that can be measured simultaneously. Through the use of Boolean gating, it is possible to determine the polyfunctionality of individual cells within phenotypically defined T-cell subpopulations. According to recently published findings in the vaccine field, the frequency of antigen-driven polyfunctional T cells may be closely linked to the effectiveness of the vaccine or the control of viral replication (22–24). While this high level of analytic sophistication permits indepth characterization of both the quantitative and qualitative aspects of vaccine-induced T-cell responses, the technology and associated assay procedures carry with them the added ‘‘burden’’ of extremely complex standardization, validation, and harmonization issues that are unlike those encountered with previous cellular assay platforms like the ELISPOT. Because of both the broad-based analytic power brought to bear through the polychromatic flow technology and the critical importance of the data quality being produced and analyzed, the remaining portion of this chapter will be devoted to the discussion of PFC assay standardization and data analysis.

Polychromatic Flow Cytometry Assay Standardization When analyzing immune monitoring data for clinical trial reporting, the goal is to conduct the data analysis in a manner that is timely, reproducible, and introduces the least amount of error. Implementing measures to improve overall assay efficiency is critical for meeting the high-throughput demands for vaccine development. High-throughput technology currently exists and is being used to automate sample processing and acquisition of samples in 96-well plate-based formats. The use of automation also improves overall efficiency, accuracy, and reproducibility for immune monitoring assay (25). Implementation of highly standardized/validated immune monitoring assays improves reproducibility of results and effectively reduces overall error, enabling investigators to measure even subtle changes in the immune response, such as those elicited by a vaccine, with confidence. The data in Figure 2 illustrate the value of using highly standardized assays to measure lymphocyte maturational subsets pre- and post-vaccination. For patient no. 12, a substantial increase in the CM CD8þ subset is observed post vaccination, while the N population is significantly contracted and the remaining maturational subpopulations remain remarkably consistent (Fig. 2). Without proper quality measures for this assay, the reproducibility in discriminating these subpopulations would have been low, and the higher degree of assay variability would have blurred the dramatic differences observed in the CD8þ CM and N subpopulations post-vaccination. Important keys to assay standardization include careful qualification of reagent performance (26–28), instrument performance, and data analysis.

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Figure 2 Two pie charts for CD8þ T lymphocyte maturational subsets, pre and post vaccination (V1 and V5, respectively) from one donor (no. 12). Maturational subsets are denoted as N, CM, EM, TE, and E. In this example, after vaccination, there is a substantial increase in the CD8þ CM subset, while the CD8þ N population is significantly contracted. Subpopulations of EM, TE, and E remain unchanged. Abbreviations: N, naı¨ve; CM, central memory; EM, effector memory; TE, terminal effector; E, effector.

Instrument Performance The goal of standardizing instrumentation is to ensure that reproducible measurements, with minimal variation, are reported over time, across batches, studies, and laboratories. Reproducible measurements of subtle changes in the immune response, such as those induced by vaccination, often challenge the limits of the instrumentation. Multilaboratory standardization and proficiency testing studies have demonstrated that a high degree, as much as 43%, of inter- and intralaboratory variability for flow-based assays may be directly attributed to instrument performance, calibration (also called setup), and data analysis (29–33). The basis for monitoring vaccine-elicited immune responses by flow cytometry is the ability to accurately classify and enumerate extremely rare subpopulations of CD4þ and CD8þ T cells on the basis of their fluorescence intensities and frequencies, respectively. With appropriate controls, flow cytometry measures may be reported in comparable units across all instruments, allowing more reproducible measures of fluorescence and more accurate comparisons for determination of positive fluorescence boundaries (34,35). This standardization eliminates arbitrary identification of populations on the basis of an operator’s perceived limits of positive fluorescence or visual analysis of data, such as commonly done when identifying CD25hi regulatory CD4 T-cells (Tregs), for example. With proper validation, optimization, calibration, and standardization, fluorescence intensity measures may be reported in stoichiometric or quantitative units (34,35). With the increase in flow cytometry laboratories performing immune monitoring assays in support of clinical trials, a number of efforts have been made to standardize measures of flow cytometry performance (34–36). A recently published method thoroughly addressed the key parameters of validation, optimization, calibration, and standardization for a flow cytometer. According to these procedures, graphs for each photomultiplier tube (PMT) are generated and analyzed to determine the optimal voltage ranges on the basis of linearity, CV, signal-to-noise, and lowest voltage (36). Optimal voltage

values (target channels) are subsequently identified by ensuring that the primary fluorescence (specific fluorescence for a given single-stained antibody-capture bead) measured in its respective PMT is greater than the secondary fluorescence (spillover into all neighboring PMTs) (36). Inclusion of a reference standard at the optimal voltage setting for each respective PMT determines the specific fluorescence or target channel values for daily calibration procedures. Calibration is performed using a fluorescence standard to set the instrument voltages back to the previously optimized target channel values, ensuring that positive fluorescence values fall in the exact same channels over time. Standardization is achieved by using the optimal target channel values over time and is a necessity for comparing samples, assays, or protocols over time. Reproducibility may be further assessed by recording repeated instrument performance measures across days and operators. All performance values measured should be recorded and monitored over time to ensure optimal performance is maintained. Equally important to the standardization of fluorescence measures is the ability to accurately and reproducibly count cell populations and report units of cell number/mL. This measurement requires stable and carefully controlled flow rates. There are a number of commercially available standards for use in determining cell number/mL; however, until recently there was no gold standard for accurately assessing counting performance. A panel of stabilized whole blood, with varying numbers of CD4, has been thoroughly tested and characterized for use in assessing linearity and limits of counting. This panel will soon be incorporated into proficiency panels, with specified values for acceptable performance, and will prove an invaluable tool for establishing accurate and reproducible counts across flow cytometry laboratories (37) Compensation and Transformation Compensation and transformation are essential elements of analyzing digital data. Most laboratories utilize antibody capture beads to generate compensation matrices. After applying a compensation matrix to a given set of data, it is wise to visually inspect the data and compare all combinations of fluorophores. Errors in compensation occur and manual adjustments to the software-defined compensation are often needed, even when careful attention has been given to ensuring that fluorescence from a compensation control is equal to or brighter than fluorescence on test samples. For digital data, logarithmic conversions, compensation, and transformations are all calculated mathematically by software. Transformations allow the low channel values to be displayed linearly, while the higher values remain logarithmic; transformation calculations are normally initiated by an operator viewing a 2D dot. The mathematical transformations for the 2D dot plot are then applied to all dimensions in the data set. Because of the instability of these calculations, a small error in compensation has the potential to be exponentially amplified throughout all parameters in the data (38,39). This type of error happens more commonly than people are generally aware and may go unnoticed, with serious consequences if operators do not routinely perform compensation checks. Until an acceptable solution is found and implemented, transformations should only be used with great care. In routine testing of clinical trial specimens, it is highly recommended that transformations should be limited to use for generating publication graphics and avoided on a more routine basis.

Chapter 20: Standardization and High-Throughput Measurement Table 2 Comparison of Assay Complexity and Data Requirements Across 4-, 9-, and 11-color Assays

Panel description #Colors #Gating generations #End points/sample Average file size/tube (mb) Average size/sample (mb) Annual storage in TB (based on 6003 tubes/yr)

Maturation and activation

Maturation and quantitative activation

Maturation and intracellular staining

4 3 18 0.20

9 6 149 20.37

12 8 1440 91.86

1.20

40.74

367.44

0.01

0.24

2.21

DATA MANAGEMENT, STANDARDIZATION, AND ANALYTIC TOOLS PFC is being used increasingly to evaluate extremely rare events in immune monitoring for clinical trials. Analysis of PFC data is highly complex, primarily due to the inherent multidimensionality of the data. By far, the biggest challenge for using PFC data in clinical trials is the difficulty of putting the data into meaningful summaries or manipulating extensively

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large data sets (beyond the limits of Excel) into a format that is reportable to statistical centers for metadata analysis of the protocol data (40,41). Using the example in Figure 1, the total number of gates for one FCS file would be 215, including 160 booleans, 40 cytokines, 10 maturational gates, 2 basic subset gates, and 3 remaining gates (scatter, viable T cells, and singlets). For an intracellular staining (ICS) assay, the minimum number of stimulations per sample is usually 3: one positive control (usually polyclonal activator such as staphylococcal enterotoxin B [SEB] or anti-CD3/anti-CD28), one negative control (CoStim, containing all reagents included in the test antigen except specific antigen), and one test antigen that is usually a mix of peptides. With three stimulations, one sample will thus result in a total of 645 measures for frequency alone; if absolute counts are also desired, the total number of measures quickly approaches 1290 for a single sample. Table 2 compares gating and data summaries for each of three different panels: 4-color Maturation/Activation panel (acquired using a FACSCalibur, an analogue system), 9-color Maturation/Quantitative Activation panel, and a 12-color Maturation/ICS panel, with gating similar to the one described above (9- and 12-color acquired on an LSRII, digital system). As the number of colors increase, the gating complexity increases, and the number of end points increase exponentially. PFC data files are extremely large due to their digital nature and high dimensionality; on average 9- to 12-color panels will result in

Figure 3 Adjusting gates by backgating on cytokine-positive events is used to increase sensitivity of the assay. After all gates are in place, a backgate is generated using cytokine-positive cells. The backgated events are then used to adjust gates so that maximum signal is detected for functional markers, while minimal negative or irrelevant events are introduced. Gates in panel A are before, during, and after backgating, respectively. The CD3 AmCyan versus Exclusion channel dot plot with data from the same file (batch control stimulated with CMVpp65 peptide mix). Before backgating (far left) a visual gate has been placed between the natural boundaries created by the density plot display. Backgating IFN-g (middle plot) illustrates how density plots might be misleading, since in this case, the cytokine-producing cells are located to the left of the gate drawn using the visual boundaries of density displays. The arrow is pointing to the IFN-g cells that fall outside of the visual gate. After backgating (far right), the left boundaries of the gate have been adjusted to maximize cytokine-producing cells within the gate (recovery) and minimize background (data not shown). The risk in moving the left boundaries of the gate is to include cells of noninterest, resulting in a reduced purity of the gate. CD4þ and CD8þ cells producing IFN-g in response to CMVpp65 stimulation are shown in panel B, before and after backgating, respectively. Backgating increased the frequency of CMVpp65-specific cells measured for both subsets.

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20 to 90 mb FCS files, respectively. Depending on the demand for PFC, an immune monitoring laboratory might expect to generate 2 to 5 terabytes of data per year. GCLP compliance requires duplication of data, meaning that an immune monitoring laboratory might expect to store approximately 4 to 10 terabytes of data per year. A need to analyze, annotate, and organize large volumes of high-dimensional data, combined with the high degree of variability associated with manual analysis, are the primary reasons that a number of efforts have recently been geared toward developing automated analysis software and data warehousing tools (41–46). Current gating software technology has not kept pace with the multidimensionality of PFC data. Existing gates are drawn using a series of sequentially gated 2-D dot plots. Nonetheless, to perform standardized analysis for immune monitoring data using conventional gating software, a number of controls and tools should be carefully employed for setting boundaries for positive fluorescence. Standard controls include negative stimulation, fluorescence minus one (FMO), and isotypes (47,48); additionally, a compromise between FMOs and isotypes, termed ‘‘gating control,’’ may prove equally useful. A gating control includes all markers used, but has isotype control antibodies for specific test antibodies, such as IFN-g for an ICS assay or CD38 for an activation assay. In addition to these standard tools for analysis, two essential tools are often overlooked in polychromatic assays: repetitive value checks and backgating. Repetitive value measures are generated from replicate determinations in a panel and are used to ensure reproducibility between tubes (49,50). Replicate determinations are made for a given sample, such as three separate measures for CD3. Using the assay in Figure 1 as an example, one patient might be stimulated for three conditions (negative control, positive control, and a specific antigen) and each of the stimulations is stained with the exact same panel of markers. Repetitive values would include 3 separate measures each for the following markers: CD3, CD4, CD8, CD45RO, CD27, and CD57 as internal measures of quality. One requirement for using repetitive values is adherence to an established acceptable range or pass/fail criteria, generally within 2%. Backgating is a means of ensuring purity and recovery for the population of interest for each respective gate in the gating hierarchy. Gates drawn on density plots using backgates appear nonconventional to an eye that is trained to visually draw gates on the basis of density; it is important to understand the concept that how data are displayed may greatly impact visual analysis of data (Fig. 3). For the example in Figure 3, backgating increased the sensitivity of the assay by increasing the frequency of CMVpp65-specific cells measured. Time is the single largest component of costs, approaching 80% for highly standardized assays using manual gating, primarily because of the requirements for standardizing manual analysis and checking data for accuracy and consistency. Any software that automates the analysis of a PFC assay with little or no operator input, would result in a significant cost savings, and would be well worth the investment, especially if there is no loss of reproducibility. In addition, displaying data in 2D dot plots fails to establish or illustrate the relationship of the populations to one another in multidimensional sample space. An ideal software would be one that uses all parameters in a given file to automatically identify and cluster subsets multidimensionally, using all of the parameters in the data file. Recent efforts have been made to automate analysis of PFC data, and some progress has been made toward automating

Figure 4 Comparison of manual gating by an expert operator versus automated cluster gating. The automated cluster gating, in addition to clustering subsets, includes a statistical inference comparison between the CoStim (negative) and the CMVpp65 peptide mix test antigen. Data shown in the right column cluster as CD4þ and CD8þ T cells that are also statistically different from the negative control staining tube. Statistical analysis was performed by Dr. Cliburn Chan (Duke Center for AIDS Research), using software developed in the Bioinformatics and Biostatistical Core. Manual gating of the same data file by an expert operator was unable to effectively remove all noise from the data (seen as a remnants of a diagonal line of nonspecific binding) and obtained slightly lower frequencies of responding cells as a result. Data were generated as a result of laboratory participation in the DAIDS Intracellular Cytokine Staining Quality Assurance Program.

multidimensional clustering that takes advantage of the high dimensionality of data (41–46). The dot plots in Figure 4 compare expert manual gating with an automated statistical analysis that includes Bayesian modeling, clustering, and inference. The automated gating was able to effectively remove a diagonal streak of background that is only partially removed by 2D manual gating. As a result, the automated cluster gating yields higher frequencies for cytokine-producing cells.

SUMMARY Innovative preventive and therapeutic vaccine strategies require highly sophisticated assay technologies capable of measuring immune responses at the single-cell level. Such immune analyses play an important role in evolving clinical immune monitoring strategies and help to determine correlates of protective immunity or ‘‘biomarkers’’ of therapeutic benefit. Of the possible assay technologies available for these purposes,

Chapter 20: Standardization and High-Throughput Measurement

only two platforms have undergone the requisite rigors of standardization and/or validation necessary for monitoring vaccine-induced T-cell responses in the context of phase I/II clinical trials. The highly quantitative IFN-g ELISPOT assay serves as a mainstay immunogenicity measure for many T-cellbased vaccine strategies. More attention has recently turned toward a more qualitative assessment of vaccine-induced T-cell responses at the single-cell level, PFC. This analysis has rapidly become the platform of choice for many ongoing and planned future trials. Although both methods have been adapted to 96well formats for high throughput, their most attractive feature is their extensive standardization resulting in the very high levels of reproducibility that translate into important tools for comparing the qualitative and qualitative immunogenicities of different vaccine strategies evaluated over numerous independent trials. The inclusion of new assay technologies in clinical trials will, of necessity, carry the critical requirement of comprehensive standardization/validation required for GCLP compliance.

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21 Transition to High-Throughput Laboratory Assays to Evaluate Multivalent Vaccines Sandra Romero-Steiner and George M. Carlone Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

Helen Baxendale and David Goldblatt Institute of Child Health, London, U.K.

INTRODUCTION The successes of the eradication of smallpox and the near eradication of polio have demonstrated that vaccination is a great tool for prevention of disease and control of epidemics and endemic disease (1,2). In the last 50 years, the number of new licensed vaccines has grown rapidly. Advances in technology and the use of reverse genetics are facilitating antigen discovery for many pathogens. However, as microorganisms evolve and adapt, vaccines must be modified to meet new challenges. There has also been a trend to an increase in vaccine antigens from single to multiple or combination antigens in a formulation. As new vaccines are developed, laboratory tools are needed to evaluate higher numbers and types of antigens and (for conjugate vaccines) to measure antibody (Ab) responses to carrier proteins such as tetanus toxoid, diphtheria cross-reactive material (CRM)197, meningococcal outer-membrane protein (OMP), or Haemophilus protein D, which can elicit protective responses upon vaccination. Thus, there is a growing demand for standardized high-throughput assays. In this chapter, we discuss the development of some multivalent vaccines using Haemophilus influenzae and Streptococcus pneumoniae as examples. We also discuss the use of surrogates and correlates of protection in the laboratory using standardized and validated assays to facilitate the licensure of new vaccines. These assays are in high demand because they are often used in conjunction with large clinical trials for vaccine evaluation. Although much effort has been placed in the standardization of assays that measure quantity of Abs, Ab function (biological activity) is a highly important component of vaccine evaluation. Bioassays must meet rigorous demands for standardization, validation, and rapid throughput.

RAPID VACCINE DEVELOPMENT AND MULTIVALENT TRENDS Rapid vaccine development and multivalent trends such as multiple serotype formulations and combination vaccines have increased the need for high-throughput assays for vaccine evaluation. For most vaccines, development and evaluation are a lengthy process. At least 10 years are needed for a new antigen to complete all safety, immunogenicity, and efficacy requirements to become a licensed vaccine. The process is more

complicated if the new product has to be evaluated as noninferior to an existing product. However, despite the lengthy and expensive process, new vaccines for Haemophilus influenzae type b (Hib), pneumococcus (Pnc), meningococcus (Men), acellular pertussis, inactivated polio, hepatitis A, chicken pox, rotavirus, and human papilloma virus have recently tracked from discovery to licensure. For example, since the 1980s, there has been a rapid development of new Hib vaccines. In the case of Hib, a single polysaccharide (Ps) antigen (polyribosylribitol phosphate, PRP) can be modified into different formulations (3). The initial formulation used pure Ps. However, immunogenicity in infants was poor, leading to the discovery that the Ps was not being recognized in infants. The antigen was then modified to add a protein component that would alter the T cell–independent antigen (pure Ps) into a T cell–dependent antigen by conjugation of the Ps with a protein carrier. This was the first use of a protein carrier for Ps. Since then many formulations were generated with different carriers (tetanus toxoid, CRM197, and outer membrane protein [OMP]). These different carriers have different presentations of the antigen; for example, when Hib PRP is conjugated to the OMP of Meningococcus group B, it elicits an early immune response to Hib at two months of age with just one single dose (4). Since then many combination vaccines that include Hib have been and are being developed (5). For example, the bivalent Hib-HepB and the tetravalent combinations including DTP and PRP-T are widely used (5), a more recent pentavelent formulation includes DTaP, PRP-T and inactivated polio (6), and hexavalent formulations that include the later antigens and hepatitis B are being evaluated (7–9). All antigens in the vaccine as well as the carrier proteins need to be evaluated, making laboratory testing highly demanding for combination vaccines (9). Advantages of combination vaccines include fewer injections per child and potential to improve routine vaccination. However, licensing new combination vaccines requires highly demanding clinical trials that demonstrate noninferiority to other products and noninterference with other antigens in the vaccine or with other vaccines that are coadministered (6,7). Like Hib, Pnc vaccines started as Ps formulations (10) which later were conjugated to protein carriers (11,12). However, for infant populations, Pnc vaccines have to adapt to the rapid change in the evolution of Pnc and the potential vaccine

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replacement with serotypes not included in the initial sevenvalent conjugate vaccine formulation, such as serotypes 19A and 6A (13,14). Also, Pnc vaccines need to meet the demands of highly diverse high-risk groups (including infants in developing countries where serotypes 1 and 5 are particularly important) and also special groups of older children and adults who are at risk. The need for additional serotype coverage has prompted the development of 10- and 13-valent formulations, despite the successful use of the licensed 7-valent formulation (PrevnarTM, Wyeth-Lederle vaccines).

VACCINE EVALUATION The complexity of efficacy trials can also increase as formulations with more antigens are evaluated. The gold standard method for vaccine evaluation is through a phase III efficacy trial with a control or placebo formulation as part of the evaluation. Using correlates of protection can facilitate vaccine licensure of vaccines against diseases where the burden of disease is difficult to estimate or when a licensed product is already available and efficacy trials with placebo controls are not ethically possible. Efficacy trials are not required in some cases because there is enough preexisting data on efficacy and appropriate correlates of protection that can be used in vitro to measure immunogenicity (3,15). For example, in 1993, the current Hib vaccine (PRP-T) (ActHibTM, Sanofi Pasteur, Pennsylvania, U.S.) was licensed in the United States based on immunogenicity, persistence of the immune response, induction of memory response, isotype and immunoglobulin G (IgG) subclass distribution, and functional Ab activity (3). Another example is the meningococcal group C vaccine (MenC-(CRM)197) introduced in the United Kingdom in a phased approach in infants to 24-year-old adults (1999–2001), which was licensed on the basis of immunogenicity rather than clinical efficacy trials (16).

SURROGATES AND CORRELATES OF PROTECTION Correlates of protection are measurable biomarkers that correlate with the protective effect of a vaccine in a target population, while surrogates are indicators of protection that can substitute for the true correlate (15). Surrogates can be either laboratory or non-laboratory measurements. For example, x-rays can be used to predict efficacy of a Pnc or Hib vaccine against pneumonia. The finding of fewer X rays showing pneumonia in vaccinated children than that in unvaccinated children would be an indicator that a Hib or Pnc vaccine is working in the target population (17). The problem is the difficulty in interpretation of X rays. Despite the efforts of the World Health Organization (WHO) to standardize the reading and interpretation of X rays to diagnose pneumonia, this surrogate of protection cannot achieve the same levels of specificity, standardization, and reproducibility of laboratorybased immunological assays. Correlates of protection are not always perfect estimates and breakthrough cases can occur (15). Some correlates are based on older studies measuring one single parameter or are derived from studies in populations living in other countries. The surrogate of protection for Hib vaccines has not changed since the licensure of the first Ps vaccine and is used to facilitate the licensure of new Hib vaccine formulations (18). Based on

immunogenicity studies using radioimmunoassay to detect antibodies to PRP in Finnish children vaccinated with a single dose of Hib PRP and a population of unimmunized adults and neonates, a minimum circulating concentration of 0.15 mg/ml of anti-PRP Ab was found to protect against invasive disease in both the vaccinated population and non-vaccinated population. However, at least 1 mg/ml of antiPRP antibody must be present in 80% of the vaccinated population between 12 and 17 months of age for protection against disease (18). This higher level discriminates between the vaccinated and non-vaccinated population later in life (older infants). This study allowed for the establishment of the minimum concentration that must be achieved in the target population to consider the population protected at that point in time (short term surrogate of protection). Since antibodies have a half life and concentrations decline unless there is a new encounter with the antigen, a long term-surrogate was established (
1 mg/ml). If the majority of the population achieves higher concentrations, protection for a longer period of time is expected. Thus, these thresholds are used for Hib vaccine licensure. The concentration of circulating antibodies required to protect against nasopharyngeal colonization is thought to be higher (
5 mg/ml) (19). Since this higher concentration protects against colonization it also leads to increased herd immunity. For Pnc the minimal concentration of type-specific Abs that must be achieved is in the range of 0.2 to 0.35 mg/mL to protect infants against invasive Pnc disease with a given serotype (20,21). Higher concentrations may be needed for protection against other forms of disease like acute otitis media and/or nasopharyngeal colonization (22–24). Exceptions to the correlate can also be found when we look at individuals. For example, a child may have antibodies (Abs) above a minimum level of protection and yet succumb to disease (25).

MEASUREMENT OF CORRELATES OF PROTECTION Techniques to measure correlates of protection are often complex and difficult to standardize. In addition, the identification of appropriate correlates is best achieved if immunogenicity studies are performed during vaccine efficacy trials (21). In some cases, identification of the correlates has not been possible. For example, despite many efforts, correlates of protection for pertussis vaccines have been difficult to establish (8,26). It has been difficult to identify among the various markers measured for pertussis which one is the true correlate of protection. In addition, standardization in assay techniques is lacking there. A common marker of vaccine induced protection used is the measurement of Abs above a minimum threshold level that has been associated with vaccine induced protection. The concentrations given above for Hib and Pnc are examples of this type of correlate. However, Abs measured in binding assays such as ELISA do not always correlate with protection and some high-risk groups (e.g., Pnc Ab in the elderly) with concentrations above these minimum levels prior to vaccination but with low functional Ab after vaccination (27). The thresholds were established for infants receiving a complete vaccination regimen and for different populations at risk, the minimum concentrations using this correlate, still need to be identified. Assays that measure a function of the Abs rather than a total Ab concentration are more likely to be better correlates of protection because they measure a biological

Chapter 21: Transition to High-Throughput Laboratory Assays to Evaluate Multivalent Vaccines

activity that correlates with protection. Assays such as the serum bactericidal activity (SBA) for gram-negative bacteria like Neisseria meningitidis and Hib have been associated with protection (28,29). Similarly, opsonophagocytosis has been associated with protection against Pnc (21,30), and neutralizing Abs have been associated with protection against viruses such as polio and yellow fever (22,31). Although measurement of a biological function tends to be a better indicator of protection (29,30), these assays are harder to perform and standardize, making reproducibility and high throughput a challenging endeavor. A new category of assays that includes indicators of immunological memory (the capacity to recall the immune response from vaccination earlier in life) is being considered as potential correlates of protection for later stages of life when circulating Abs have waned. Examples of these correlates are avidity assays, which are modified ELISAs that measure the strength of the antigen-Ab reaction, and bioassays such as the enzyme-linked immunosorbent spot (ELISPOT), which measure the number of Ab-producing B cells in the peripheral blood in response to a specific antigen. While Ab measurements (both concentration and functional titer) have proved useful in assessing the effectiveness of vaccine priming for quality memory responses, cellular studies have demonstrated that B-cell memory may persist in the absence of detectable Ab (32,33). These studies suggest that memory B-cell assays may provide a more reliable index of persistent memory than standard Ab ELISAs and may be used to determine whether booster immunization in the absence of detectable serum Ab is really required to maintain immune protection (34,35). Therefore, standardized and reproducible ELISPOT assays are needed. Only assays that have been standardized and validated can be used in immunogenicity studies that support the licensure of new vaccines. Assay standardization is an absolute requirement to be able to compare the results generated in different laboratories, after different formulations, or if different lots are used. An example of an assay that has been successfully used by industry, because of the level of standardization and validation, is the ELISA assay to measure IgG Abs to Pnc Ps antigens. For this Pnc ELISA, there is a consensus protocol (36) that was generated after two large multilaboratory assay comparisons (37). There is a set of 12 quality control sera that have WHO assignments to help new laboratories establish the ELISA protocol and a reference standard serum (89SF, available at the FDA) with type-specific Ab assignments for IgM, IgG, and IgA. A new reference serum (007SP) is currently being generated by M. Blake at the Food and Drug Administration, United States. The new standard will undergo similar characterization to the 89SF and will serve as a reference material for both functional and nonfunctional assays. Another example of assay standardization and validation is the opsonophagocytosis assay (OPA) that measures the capacity of the Ab to kill bacteria (i.e., Pnc) in the presence of complement and phagocytic cells (38). The concept of phagocytosis has been around for more than 100 years since the first observations by Metchnikoff (38). However, the capacity to perform this assay in a reproducible and standardized format for Pnc vaccine evaluation was not possible until in vitro phagocytic cells were introduced into the assay, which is now performed in a microtiter plate format (39). This standardized assay has undergone a number of modifications and validations so that it can now be applied under the strict requirements of quality

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Figure 1 OPA measures functional antibodies to Pnc by determining the serum dilution with 50% killing of the bacteria in the complement control wells (39). The standard OPA assay has been used in most of the large vaccine clinical trials (38). This assay has been multiplexed by using a mixture of antibiotic resistant strains and replica plating in selective media to measure killing titers in panels of four or seven serotypes, increasing the throughput of the assay (42–44). Colonies are counted with an automated counter or with a fluorometer (for the metabolic activity of the surviving bacteria). Data is automatically analyzed to determine either continuous or discontinuous titers (42,44).

management systems by vaccine manufacturers (40,41) (Fig. 1). Currently, the assay is available in a multiplex format with up to seven different serotypes that can be evaluated at once (see sect. High-Throughput Assays That Measure Antibody Function). Assays that measure immunological memory such as avidity and ELISPOT have also reached a high level of standardization and validation, and they have been successfully implemented in reference laboratories. Over the last 20 years, there have been various modifications of the ELISPOT assay originally described by Czerkinsky et al. in 1983 (45) to be able to standardize and improve the throughput of this bioassay. Originally, the substrate was added to liquid agar, which solidified on the ELISPOT plates to permit spot count. More recently, a variety of membranes have been successfully used to quantify antigen-specific Ab-secreting cells (AbSCs) to both proteins and Ps vaccine antigens (46,47). With the increasing number of antigens in a formulation, assays that are correlates of protection also need to have a high throughput. Industry has addressed this need by having many operators perform the same test or parts of a test, or by introducing robotics and automation when possible. However, new assays have been developed that can perform multiple assay determinations in the same reaction mixture, therefore reducing the number of operators needed and/or the assay time. For example, an immunogenicity trial with 5000 samples

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for 7 different antigens (35,000 assays) with a single operator performing 10 ELISA plates (5 sera per plate) a day for 5 days a week would take 140 weeks (2.6 years) to be completed. If the same antigens are tested simultaneously, the same operator can complete the study in 20 weeks (0.4 years).

HIGH-THROUGHPUT ASSAYS THAT MEASURE ANTIBODY QUANTITY In this section, we discuss specific examples of high-throughput assays that can be found in the evaluation of Abs to Men and Pnc Ps antigens (Table 1). For example, for IgG Ab concentrations, microsphere-based assays can measure simultaneously the Ab to different antigens. One of these technologies uses the LuminexTM (Austin, Texas, U.S.) format for capturing mean fluorescence units that can be converted into Ab concentrations if a reference serum with Ab assignments or a calibrator is available. Initially, this technique was widely used for the measurement of cytokine concentrations (58). Since then, several assays have been developed for the evaluation of Pnc Ab, with various degrees of success in terms of validation. One of the first assays described for Pnc antigens was described by Pickering et al. (48) with a companion flow cytometric assay for the quantitation of Ab to tetanus, diphtheria, and Hib (59). Biagini et al. described the covalent linkage of each of 23 Pnc Ps to amino groups in the microspheres using sodium periodate to oxidize the Ps (49). However, this assay requires great care in the oxidation step, with periodate making this technique difficult to implement in a reproducible manner. A modification of this assay is currently being evaluated at the Centers for Disease Control and Prevention (CDC) in the United States (60). Schlottmann et al. described a modified assay that uses Ps

conjugation via the carboxyl functional groups in the microspheres using DMTMM (4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4methyl-morpholinium) (52). The nanoplex assay developed by Lal et al., which has been validated at the Health Protection Agency in the United Kingdom (51), uses a modification of the poly-L-lysine conjugation technique described by Pickering (48). The multiplex assay gave high limits of detection (between 32.3 pg/mL for serotype 1 and 109.7 pg/mL for serotype 19F) and correlated well with ELISA-derived Ab concentrations with r values between 0.95 and 0.98. This assay has currently been validated for the measurement of 12 different anticapsular Abs to Pnc and has outperformed other microsphere-based assays in an interlaboratory comparison (54). The nanoplex assay has the capacity of being combined in a 13-plex format along with a tetraplex assay for quantitation of Abs to Men Ps, A, C, Y, and W135 (50). This multiplex assay can reduce the amount of sera needed for the evaluation of both meningococcal and pneumococcal antibodies. Another example of multiplex determinations of Ab to Pnc anticapsular Ps is the electrochemiluminescent assays developed and standardized by Marchese et al. (55) using Meso Scale Discovery (MSDTM) technology. This technology allows for multiplex ELISA determinations with a high degree of reproducibility. This novel technique uses carbon-coated electroplates, which do not require a chemical modification of the Ps for antigen coating. In general, multiplex assays greatly reduce wastage of materials, the amount of serum sample, reagents, and operator time while increasing the throughput of assay determinations by a single operator and the limits of detection (Table 1). Some drawbacks of these technologies are the cost of more sophisticated instruments such as flow cytometers and electrochemiluminescent readers that require

Table 1 Multiplex Assays for Measuring Specific Antibodies to Neisseria Meningitidis or Streptococcus Pneumoniae Assay/method IgG/Luminex Microspheres IgG/Luminex Microspheres IgG/Luminex Microspheres IgG/Luminex Microspheres IgG/Luminex Microspheres IgG/XMAP IgG/FlowAps IgG/MSD Electro plates OPA/Fluorospheres OPA/Fluorospheres/ Bact. OPKA/ Anti-microbials OPKA/Fluorescence OPKA/ Anti-microbials a

Bacterial antigens

Single-plex correlation

Limit of detection

Standardized / validated

Clinical trials use

Pnc 14 types

r values 0.92–0.98

NA

Yes/Yes

Yes

Pnc 23 types

Evaluated only 89S-2 r values 0.86–0.97

20–600 pg/mL

Yes/No

No

260–650 pg/mL

Yes/Yes

Yes

r values 0.95–0.98

32–110 pg/mL

Yes/Yes

Yes

Men groups A, C, Y, W135 Pnc 9 types Pnc 12–15 types Pnc/7–15 types Pnc/7-22 types

NA

0.6–53 ng/mL

Yes/Yes

NA

r
0.931a r ¼ 0.992a

NA NA

Yes/No Yes/No

No No

Pnc 8 types

r ¼ 0.994

8–66 pg/mL

Yes/Yes

No

Men groups A, C, Y, W135 Pnc, panels of 3–4 for 7 types Pnc 7 types

Mean rSBA = 0.96

Titer of 8

Yes/Yes

Yes

r values 0.61–0.91

Titer of 8

Yes/Yes

Yes

NA

Titer of 10

Yes/No

NA

Pnc 7 types

r values 0.76–0.97

Titer of 8

Yes/Yes

Limited

Pnc, panels of 4 for 13 types

r
0.97

Titer of 4

Yes/Yes

Yes

Compared with Lal et al. (51), not to a single-plex ELISA. ELISA QC panel concordance was 42% to 55% (54).

Reference/year Pickering et al., 2002 (48) Biagini et al., 2003 (49) Lal et al., 2004 (50) Lal et al., 2005 (51) Schlottmann et al., 2006 (52) 53 Whaley et al. 2008 (54) Marchese et al., 2008 (55) Martinez et al., 2002 (56) Martinez et al., 2006 (57) Bogaert et al., 2004 (43) Bieging et al., 2005 (44) Burton et al., 2006 (42)

Chapter 21: Transition to High-Throughput Laboratory Assays to Evaluate Multivalent Vaccines

expensive maintenance contracts. Also, these assays require costly reagents like microspheres of precise diameter with premixed fluorescent markers or specialized reaction plates, which need to be spotted by the manufacturer using nanoparticle robotics equipment. These types of materials cannot be produced by the testing laboratory and must be purchased from the manufacturers.

HIGH-THROUGHPUT ASSAYS THAT MEASURE ANTIBODY FUNCTION Like Ab quantitative assays, the multiplex measurement of functional opsonophagocytic activity of Ab to different Pnc types has been developed, standardized, and implemented in various clinical trials with good success (38) (Table 1). The assay described by R. Burton and M. Nahm, MOPA4, allows for the measurement of OPA titers to panels of 4 antimicrobial resistant strains that cover 13 different Pnc serotypes (42). Each serotype can be differentially selected in this killing OPA (OPKA) using growth medium containing the appropriate antimicrobial, and the colonies can be counted with an automated colony counter. This assay has a high degree of agreement (r values
0.97), with the standard assay performed in a single-plex format. A modification of this assay was described by Bogaert et al. for seven pneumococcal serotypes (43). Similarly, fluorescent multivalent OPAs measure the OPA titers to seven different serotypes by means of a fluorescent metabolic indicator (Alamar BlueTM), which correlates with the viable count of Pnc after OPA (44). This assay has the capacity to measure functional OPA titers without having to count colony-forming units and has a good correlation (r values 0.76–0.97) with the standard OPA. Flow cytometric OPAs that measure the uptake of fluorescent microspheres coated with Ps or fluorescently labeled bacteria have also been used for Pnc (57) and Men (56). This assay format demonstrates a good level of correlation with the standard Pnc OPA (r values 0.68–0.92) or with Men SBA (r value ¼ 0.96). Microsphere-based assays offer the ability to bind different types of antigens (Ps, proteins, peptides, etc.) for evaluation of the immune response. The major criticism to this type of assays is that they are too far removed from the pathogen itself, since a single component is used in the reaction and the live microorganism is not being used. A minimum OPA titer of 1:8 has been identified as the amount of circulating functional Ab at which protection against invasive disease is expected in the vaccinated host (21). However, higher titers may be needed for protection against acute otitis media (23). Since the trend is toward multiplex OPAs, there is a need for highly standardized and validated multiplex assays that can accurately measure functional Ab at these low levels (42). Although there are a number of assays currently available, they are all at different stages of development, evaluation, validation, and adoption. One lesson to be learned from the measurement of Pnc correlates of protection is that the multivalent nature of the vaccine formulations has forced a change in the technologies to meet the overwhelming demand for high-throughput testing in the laboratory.

ELISPOT ASSAYS THAT MEASURE IMMUNOLOGICAL MEMORY For some current vaccines, the immunization schedule relies on induction of memory responses based on the administration of

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only three doses in early infancy and no reinforcing dose in the second year of life (61). In its original form, the ELISPOT assay was used to quantify circulating AbSCs to vaccine antigens within the small window of time postimmunization (7–14 days) during which circulating antigen-specific AbSC could be detected in peripheral B memory cells (PBMCs) isolated from whole blood. The limits of detection of a specific AbSC using this assay were estimated to be 1 in 100,000 PBMCs. This window is thought to represent the time when, following parenteral immunization, vaccine-primed antigen-specific memory B cells and plasma cells migrating from the spleen to the bone marrow peak in the peripheral blood. After this time, antigen-specific memory B cells continue to circulate in low numbers, but since they are not actively secreting Abs, they are not detectable by the standard ELISPOT assay (46). Although ELISPOT is not yet a high-throughput assay, many improvements have been made to this method to make it feasible as a biomarker for the evaluation of memory in the laboratory. In the late 1990s, a marker for human memory B cells (CD27) was identified and characterized (62). This marker was then used to identify ways of preferentially activating and expanding memory B-cell numbers in vitro (63) to improve the efficiency of detection of low-frequency antigen-specific memory B cells with time post immunization (35,64). A variety of techniques have subsequently been described to expand memory B-cell numbers. One of the most effective strategies involves the inclusion of methylated bacterial oligonucleotides (CpG DNA) (65). When added to peripheral blood mononuclear cells (PBMCs) in vitro, CpG DNA binds to the toll-like receptor ligand TLR9, which is preferentially expressed on memory B cells (66,67). This induces memory B cells to divide and differentiate to pre-plasma cells/plasmablasts that are readily detectible by ELISPOT (Fig. 2). Using these assays, memory B cells to vaccine antigens have been identified from PBMCs years after immunization (35,68) and the ability of different vaccine formulations to recruit memory responses has been assessed (69). To enable the application of antigen-specific B-cell ELISPOT to comparisons across vaccines in different patient populations, these assays must be standardized so that interlaboratory comparisons may be made. This approach of quantifying lasting memory responses may then be applied to

Figure 2 Quantification of pneumococcal polysaccharide specific IgG memory cells by ELISPOT following in vitro expansion culture of PBMCs with CpG 2006 compared with nonstimulated culture (R10).

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the high-throughput assessment of vaccine responses in humans. An essential component of this standardization is to be able to work on batched frozen PBMC samples. Although the freeze-thaw cycle does introduce a number of potential biases to the analysis, the ability to detect antigen-specific memory B cells is relatively unaffected by this procedure (46). While quantification of antigen-specific IgG producing memory cell numbers has been the main objective in developing these assays, depending on the vaccine antigen, age, and immune experience of the target population, quantification of IgA and IgM AbSC number may be equally important as a measure of effective induction of B-cell memory. The IgM producing marginal-zone memory B-cell population is considered to play a particularly important role in protection against invasive Pnc disease, particularly in the absence of affinity maturation and class switching of the Ab (70,71). Quantification of this population may thus provide useful information in highrisk populations with regard to susceptibility to Pnc infection (72). Marginal-zone and IgA memory B-cell populations respond similarly to IgG producing memory AbSC in culture, so they may be expanded for quantification in the same way. Using these techniques, one may evaluate the likely relative contribution of different B-cell populations to the memory response and the effect of immunization in boosting these different populations. Such analysis may improve the understanding of the biology of protective immunity in different populations so that more accurate in vitro correlates of protective immunity are used to inform immunization strategies.

MEETING THE DEMANDS OF VACCINE EVALUATION Regulatory agencies such as the EMEA and the FDA license vaccines after a careful review of immunogenicity data generated during the various clinical trials (phase I, II, III and sometimes IV). The link to clinical efficacy is crucial for the evaluation of new vaccines lacking efficacy data. For example, correlates of protection will need to be established for the investigational Pnc protein vaccines. The development of standards with appropriate Ab assignments is easier when there is only one antigen in question; however, as the valency of the vaccines increases, the assignments to one single standard are more demanding. In the United States, the FDA has been critical in the production and availability of such reference materials. However, for worldwide availability, the WHO has established collaborating centers that can facilitate access to these reagents. In addition, to assure assay performance quality control, sera must also be available to the various testing laboratories. The scientific community working on Pnc, Men, and Hib has benefited from the availability of these reagents for assay development and for vaccine evaluation (36,73). Another element of assay standardization has been the comparability of assay methodologies across laboratories. Efforts to standardize methodologies can be demonstrated by the various interlaboratory studies performed for ELISAs, OPAs, and SBAs. Standardization of the methodologies allows for comparability of vaccine products and facilitates the licensure of new vaccine formulations using the concept of noninferiority (74). Hence, a new product can be evaluated against a licensed product to gain approval for use in the target population. Use of correlates of protection with well-defined end points can facilitate this process and speed the time for vaccine licen-

sure (15). Traditionally, quantitative assays that measure Ab concentrations have been used for this purpose because these assays are easier to standardize. However, a new trend toward using functional assays that are better correlates of protection is changing the field of vaccine evaluation to meet the demands of the regulatory agencies. Also, comparison of the reference data generated by the standardized monoplex assays helps with the development and validation of new multivalent assays that can simplify the testing of the thousands of samples that are collected in large immunogenicity clinical trials. Correlates of protection are yet to be determined for populations at risk like the elderly, HIV-infected, splenectomy, sickle-cell, and transplant patients. Much effort is being placed in the identification of correlates of protection and the development of assays that can measure function and relevant biomarkers. A new area of interest is the study of genetic signatures of immune response which may correlate with responses in the long-term after vaccination (31). These new technologies may be able to address this need in future.

SUMMARY Great advances have been made in the development of new vaccines in the last 50 years. However, the complexity of the new combination vaccines and the use of multivalent vaccine formulations require that assays used in the laboratory as correlates of protection be modified to be able to meet the demands of both industry and the approval of the regulatory agencies for licensure. Future trends indicate that more antigens are likely to be part of the routine infant immunization, making combination vaccines highly desirable. Multiplex technologies have been developed that can facilitate testing of multiple antigens in a single reaction; however, much effort is still required to improve the validation of these assays and to define correlates of protection for populations at risk for which the current ‘‘correlates’’ do not seem to accurately predict protection. New technologies that are indicators of memory or genetic markers that can predict protection by generation of immunological signatures may have a niche when studying special populations at risk.

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22 The Challenge of Vaccine Protection in Very Young Infants Claire-Anne Siegrist WHO Collaborating Centre for Neonatal Vaccinology, Departments of Pediatric and Pathology and Immunology, University of Geneva, Geneva, Switzerland

INTRODUCTION Currently used infant vaccines and schedules mostly induce protection after several vaccine doses, that is, only at several months of age. As a result of this delayed induction of protection, whooping cough still results in approximately 360,000 infant deaths annually, despite the administration of three doses of diphtheria-tetanus-pertussis vaccine to 80% of the world’s children before their first birthday (1), as infants too young to have yet completed their three-dose vaccination schedule and unvaccinated infants remain at a significant risk of pertussis (2). At a global level, more than 2.5 million infant deaths annually result from acute respiratory and diarrheal infections, many of which could be prevented by immunization with vaccines, against a relatively limited number of viral and bacterial pathogens (3). To prevent these infant deaths, vaccines and immunization strategies would have to safely induce protective responses more rapidly after birth, prior to pathogen exposure that frequently occurs in very early life. Unfortunately, infant antibody responses are also of short duration, which may result in reoccurrence of vulnerability to pathogens. The challenge, which will also have to be met by novel vaccines against major later killers, such as tuberculosis, malaria, and HIV, is thus to induce early and sustained protection despite the immaturity of the neonatal immune system and the presence of antibodies of maternal origin. The objective of this chapter is to review the current understanding of the determinants, which may either limit or support the induction and persistence of vaccine responses in neonates and young infants, with a special focus on antibody-mediated protection, and to highlight areas in which further research is needed.

CHALLENGES FOR THE INDUCTION OF ANTIBODY RESPONSES IN EARLY LIFE Limitations of Early-Life Antibody Responses Limited Infant Responses to Polysaccharide Antigens Infant and toddler responses to most bacterial capsular polysaccharides (PS) are markedly limited, which contribute to their high susceptibility to infections with encapsulated bacteria such as Haemophilus influenzae (HIB), Streptococcus pneumoniae, and Neisseria meningitidis. The same limitations affect most purified polysaccharide vaccines, which remain poorly immunogenic before the age of 2 years and exhibit an age-dependent increase in vaccine efficacy between 2 and 10 years (4). Factors that limit infant responses to PS include (i) low complement activity, which limits the deposition of C3d on bacterial PS, (ii) weak

expression of surface C3d-receptors (CD21) on infant B lymphocytes, limiting synergy between B cell receptor and complement receptor-mediated activation, and (iii) structural immaturity of the splenic marginal zone (5) to which C3dbound PS preferentially localizes in adults, and a timing coincidence between the appearance of adultlike marginal zone B cells with adult features and the acquisition of the ability to mount an immune response to PS was reported (5). Collectively, these factors limit the capacity of marginal zone B cells and B1 cells to respond rapidly to particulate bacterial antigens (Fig. 1) (6). The recognition of this limitation, intrinsic to infant and toddlers, led to the development of glycoconjugate vaccines, which attach bacterial PS to a carrier protein. Processing and presentation of carrier peptide fragments at the surface of antigen-presenting cells (APCs) recruit CD4þ T cells, which provide costimulation to infant B cells, and thus induce immunogenic and protective responses in young infants. Despite the strong immunogenicity of glycoconjugate vaccines, the magnitude of the IgG antibody responses that they elicit still depends on the age at which they are administered. For example, a single dose of HIB-conjugate vaccine elicits progressively higher serum anticapsular antibody concentrations when administered, respectively, at 2 to 3 months, 4 to 6 months, or 8 to 17 months (7), and two doses of pneumococcal conjugate vaccines are required to elicit adultlike responses in toddlers (8). Limited Early-Life Antibody Responses to Protein Antigens The magnitude of IgG antibody responses that may be elicited by protein antigens, whether in subunit or live attenuated vaccines, is also directly related to age at immunization (Table 1). The influence of immune immaturity on most currently available protein-based vaccines (such as combined diphtheria-tetanus-pertussis or hepatitis B vaccines) may not be assessed directly, as these vaccines do not induce significant antibody responses to a single vaccine dose even in adults. However, much higher antibody responses to the potent hepatitis A vaccine are elicited in toddlers than in infants of seronegative mothers (9), and the stepwise increase of antibody concentrations following measles (10–12) or mumps (13) immunization when immunization is delayed from 6 to 9, 12, or 15 months of age directly reflects the influence of age at immunization. The mechanisms that limit early-life antibody responses to protein antigens, whether included in protein, subunit, inactivated, or live attenuated vaccines, are numerous (Fig. 2) and not yet fully understood. Recent studies assessing the

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Figure 1 Limitations of marginal zone B-cell responses to bacterial polysaccharides. Bacterial PS arrive in the MZ, where they may bind to MZ B cells. These lymphocytes rapidly expand into plasma cells that essentially produce IgM with some IgG antibodies but do not differentiate into memory B cells. Antibody responses elicited in adults are of a higher magnitude and more prolonged persistence than those elicited in young children. Abbreviations: PS, polysaccharides; MZ, marginal zone.

Table 1 Characteristics of Age-Dependent Limitations of Immune Responses to Conventional Vaccines Observed in Neonates and Very Young Infants Immune responses with age-dependent limitations Magnitude of antibody responses Limited induction of IgG responses to protein Ag Shorter duration of IgG Ab responses Limited induction of IgG responses to most polysaccharides Quality of antibody responses Diminished affinity maturation of IgG antibodies Limited IgG2 responses Limitation of IgG repertoire Antigen-specific T-cell responses Limited primary IFN-g responses Limited CTL responses Limited IL-12 production by dendritic cells

Age at which mature responses are reached >12 mo >12 mo >18–24 mo >6 mo (?) >12– 18 mo >8–12 mo (?) >12 mo or later (?, V) >12 mo or later (?, V) >12 mo or later (?, V)

Schematic comparison of main characteristics, although notable exceptions may exist. In contrast with the age-dependent limitations of certain immune responses listed in the above table, the induction of memory B cells and the ability to prime T cells are already highly efficient at (or even before) birth of the full-term human infant. (?) Limited data available. (V) Major influence of antigen/vaccine type.

influence of age on antibody responses to human vaccines in neonatal, infant, and adult mice yielded observations very similar to those of human infants, providing that immunization was initiated more than seven days of age (rather than in the immediate neonatal period) to compensate for the greater immaturity of newborn mice (reviewed in Ref. 14). Studies assessing the various stages of antigen-specific B cell differentiation in these preclinical models of early-life immunization demonstrated that the weaker antibody responses reflect a delayed and weaker induction of the germinal centers (GCs) in which antibody-secreting cells (ASCs) develop (15). This likely reflects immaturity of neonatal B cells (16), CD4þ T cells, and APCs, as well as that of additional determinants of the postnatal development of lymphoid organs (17). The definition of the relative contribution of each of these factors to the limited capacity of early-life B-cell responses (Fig. 2), and the identification of strategies likely to circumvent such immaturity-associated limitations require additional studies.

Limited Duration of Early-Life Antibody Responses Another challenge for early-life immunization is that the strong antibody responses that are eventually elicited after several doses of immunogenic vaccines have been administered are of a shorter duration than the responses elicited when these vaccines are given at more than12 months of age. This decline of IgG antibodies within 6 to 12 months after immunization requires, ideally, that an additional dose of vaccine should be administered in the second year of life in order to maintain protection. Regrettably, this strategy of delivering an additional dose in the second year of life is difficult to implement in many developing and transitional countries of the world. The shorter persistence of infant IgG antibody responses reflects a limited in vivo half-life of plasma cells generated in early life, which essentially depends on their homing, establishment, and survival in the bone marrow (BM) compartment (Fig. 2). Similar limitations occurring in infant mice (15) result from a limited capacity of the BM compartment to support plasma cells’ survival (18). This was recently ascribed to the insufficient expression of a proliferation-inducing ligand (APRIL), a tumor necrosis factor (TNF) family receptor ligand, in the early-life BM compartment (19). Whether the same limitations exist in human infants requires studies that are difficult to perform. However, the demonstration that environmental (rather than genetic) factors control antibody persistence in infant twins (20) provides indirect evidence for a limited capacity of the early-life BM compartment to sustain plasma cell responses. Does Early-Life Immunization Limit the Quality of Antibody Responses? Whether early-life immunization results in antibodies of a different quality is an important question. Infant vaccines preferentially elicit IgG antibodies regardless of age at administration (21). However, infant immunization mainly elicits IgG1 and IgG3 isotypes, and IgG2 antibodies remain weak during the first 18 months of life (22) even for vaccines that induce preferential IgG2 responses in adults (Table 1). The relative capacity to induce high-avidity antibodies in early life is of significant concern, as avidity is a direct marker of functional efficacy. Infants indeed produce antibody of a significantly lower avidity (with absent bactericidal activity) following N. meningitidis infection as compared to older children and somatic hypermutation of Ig genes in infant B cells slowly increases between 2 and 10 months of age, with evidence for selection only from 6 months onward (23,24). Years ago, studies in infant mice with hapten-conjugated antigens demonstrated that the capacity to enter into an efficient antibody maturation process was age related. All together, these

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Figure 2 Limitations of early-life B-cell responses to protein antigens. Protein Ag reach the spleen/nodes by free-fluid diffusion (small molecules) or transported by DCs that have picked-up antigens at the site of injection.(1) B-cell activation results from cell-cell interactions between Ag-specific B cells, Ag-bearing DCs and Ag-specific CD4þ T helper cells (Th), under the control of regulatory T cells (Tr). (2) Activated B cells are attracted by Ag-bearing FDCs that nucleate GCs. In these GCs, signals from FDCs and Tfh drive Ag-specific B cells into a clonal expansion. (3) Within GCs, B cells receive signals that orient their differentiation toward either plasmablasts (PB)/short-lived PCs, or toward memB cells. MemB cells migrate through the blood back to the T cell zone of spleen/nodes, ready for a new round of expansion at the time of repeat exposure. (4) Antibody-producing PCs leave the spleen/nodes and home through the blood toward the BM where they receive specific survival signals from SCs. These signals support their establishment into the BM compartment and their final differentiation into longlived BM PC. The weak magnitude and shorter persistence of antibodies elicited in early life result from limitations in steps 1 to 4. Abbreviations: Ag, antigens; DCs, dendritic cells; FDCs, follicular dendritic cells; Tfh, follicular T cells; GCs, germinal centers; memB cells, memory B cells; PCs, plasma cells; BM, bone marrow; SCs, stromal cells.

observations generated concern that early-life immunization may be associated with induction of antibodies of a weaker functional efficacy. However, we recently observed that, in contrast to hapten-based vaccines, two human infant protein vaccines (tetanus and pertussis toxoids) induce an adultlike neonatal murine antibody avidity maturation process (25). In accordance, studies have demonstrated that the affinity maturation machinery is already functional in the first months of life (26). Ongoing studies in neonatally primed infants are expected to shed light on this early affinity maturation process.

Does Early Immunization Induce Neonatal Tolerance or Priming of Memory Cells? Inducing tolerance by neonatal immunization is a concern that has originated from murine studies. However, in contrast to newborn mice and their profoundly immature immune system, there are only few reports of hyporesponsiveness to subsequent

vaccine doses following neonatal immunization with whole-cell pertussis vaccines, PRP-OMPc, or N. meningitidis group C polysaccharides (MenC PS). In contrast, secondary response patterns are observed when infants who were primed in the neonatal period are then boosted at one month of age with hepatitis B or polio vaccine (reviewed in Ref. 14). Additional evidence was recently generated by the successful priming of neonates against pertussis and the induction of accelerated responses to the first infant vaccine dose (27,28). Surprisingly, neonatal administration of certain strongly immunogenic infant vaccines [such as tetanus toxoid vaccine or DTPa-based vaccines (29)] did not enhance, and in some instances even limited responses to subsequent vaccine doses. Thus, whether priming of memory cells may or not be achieved by neonatal immunization may currently not be predicted by preclinical studies. As early prime– boost immunization strategies are likely to prove essential for vaccine-mediated prevention in early life, further studies should attempt to identify the determinants of neonatal priming.

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Influence of Maternal Antibodies on Early-Life Antibody Responses It has long been recognized that residual maternal IgG antibodies (MatAb) passively transferred during gestation may inhibit infant vaccine responses to measles and oral poliomyelitis vaccines, and more recently that they may also affect responses to non-live vaccines (reviewed in Ref. 14). The main determinant of MatAb-mediated inhibition of antibody responses was identified as the titer of MatAb present at the time of immunization, or rather as the ratio between vaccine antigen and MatAb (30–32). Indeed, reducing MatAb titer at time of immunization or enhancing the dose of vaccine antigen may both circumvent the inhibition of infant antibody responses in human and murine infants. This is best explained by the fact that following introduction of a vaccine antigen into a host with preexisting passive antibodies, MatAb readily bind to specific B cell vaccine epitopes, preventing access of infant B cells to the same determinants. If the vaccine antigen/MatAb ratio is low, this prevents access of infant B cells to B-cell epitopes, and therefore inhibits their differentiation into antibody-secreting cells. At a higher ratio, some B-cell epitopes may remain unmasked by MatAb, and thus available for binding by infant B cells and priming of B-cell responses. Thus, strategies to circumvent MatAb inhibition of vaccine antibody responses currently mainly include delayed vaccine administration, awaiting decline of MatAb or use of higher vaccine doses. Whether slow-release vaccines or certain delivery systems could better shield B-cell epitopes from MatAb is an interesting possibility, which awaits confirmation. In theory, mucosal vaccines should prove better able at escaping from MatAb inhibition, as concentrations of MatAb reaching infant mucosae are significantly lower than those reaching their serum. However, this may only be the case for immune responses elicited directly at the mucosal surface and not into the draining lymph nodes where MatAb concentration is higher.

Perspectives for Enhancing Early-Life Antibody Responses In neonatal and infant murine models of immunization, certain adjuvant formulations are able to significantly enhance early-life vaccine responses, whereas others fail to do so despite their strong adjuvanticity in adult animals (reviewed in Ref. 14). In infants, coadministration of BCG at time of neonatal hepatitis B immunization strongly enhanced (50-fold) HBsAg antibody titers after the third vaccine dose compared with control infants (33), an influence likely to reflect the known maturation influence exerted by BCG on dendritic cells (DCs). Enhancing DC/T cells/B cells interaction, for example, through adjuvantation, may thus have a positive influence on the magnitude of antibody responses elicited in neonates. Whether this will enhance responses to the first vaccine dose remains to be tested. Indeed, observations gathered with a large panel of adjuvants in murine models (reviewed in Ref. 34) suggest that some limiting factors may not be corrected by enhanced DC/T cells/B cells activation. Novel antigen delivery systems, such as DNA vaccines, have not yet been tested in human neonates but were extensively studied in neonatal animal models. DNA vaccines induced similar antibody responses in newborn and adult mice (reviewed in Ref. 34) but failed to induce stronger earlylife antibody responses than those elicited by conventional protein/, subunit, or live attenuated vaccines. Accordingly, DNA immunization of newborn or infant nonhuman primates

against hepatitis B, HIV, or influenza also resulted in weak antibody responses (35), and sequential bleeding indicated lack of antibody responses prior to four or eight weeks of age, after two or three vaccine doses (36). Thus, vaccine formulations/ delivery systems capable of rapidly inducing strong antibody responses in early life have not yet been identified. This calls for a better understanding of the limiting factors, so that strategies can be designed to circumvent them. Indeed, although early priming–later boosting strategies are currently the most promising for enhancing early-life antibody responses, the time required for completion of such strategies is likely to be a limiting factor against pathogens for which exposure occurs very early in life. Alternative strategies include maternal immunization, as recently demonstrated efficient against infant influenza (37).

CHALLENGES TO THE INDUCTION OF STRONG T-CELL RESPONSES IN EARLY LIFE Characteristics of Early-Life CD4þ and CD8þ T-Cell Responses In contrast to the slow maturation of antibody responses, acquisition of antigen-specific T-cell responses is an early event. The age-dependent maturation and differentiation of Th1 (IFN-g secreting) and Th2 (IL-4, IL-5, IL-13 secreting) T-cell responses is, however, yet poorly characterized (Table 1). T-cell proliferative responses following BCG were stronger when administration was delayed from birth until two to six months of age in some studies, whereas adultlike IFN-g responses to neonatal BCG were reported in The Gambia (38). In contrast, Gambian infants showed defective IFN-g responses during the primary phase of the response to oral polio vaccine (39), as compared to adults. Analyses of T-cell responses to measles and mumps vaccines indicated similar antigen-specific T-cell proliferative and IFN-g responses in infants immunized at 6, 9, or 12 months of age, but lower infant responses than those of adult controls (13,40). Infant T cells also showed a limited capacity to increase their IFN-g release in response to IL-12 supplementation (40), which required both IL-12 and IL-15 (41). BCG vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles (42), and CD154 is not expressed at adult levels prior to the second year of life (43). A limitation of these MMR/OPV/BCG studies is that they cannot include previously unprimed naive adult controls. To precisely define the influence of immune immaturity on T cell differentiation thus awaits additional clinical evidence. Little is yet known of the maturation of human infant CD8þ cytotoxic T lymphocyte (CTL) responses. Although infection-induced CTLs may be detected within the first weeks of life, CTL responses could also be age and vaccine dependent (44–46). As an example, CTLs were recovered in infants following influenza infection, but not following immunization with a live influenza vaccine, suggesting that a certain immunogenicity threshold had only been reached in infected infants (45). Thus, it seemed likely that the maturation of CD8þ cytotoxic responses will prove age- and vaccine type–dependent in human as in mice, as supported by recent studies (47,48).

Which Are the Factors Limiting Early-Life T-Cell Responses? Studies assessing the determinants of early-life T-cell responses were long limited to murine models of early-life immunization. They indicated that antigen-specific T-cell responses may be

Chapter 22: The Challenge of Vaccine Protection in Very Young Infants

readily elicited at an early stage, but that early immunization is associated with lower IFN-g responses (and higher IL-4, IL-5, and IL-13 responses) to most conventional vaccines (reviewed in Ref. 14). Altogether, these observations suggest a preferential differentiation of early-life T-cell responses to viral/protein vaccines toward the Th2 pathway, as a ‘‘default’’ developmental pathway (reviewed in Ref. 49) reflecting suboptimal APC-T cell interactions. Evidence that neonatal APC function may be immature has indeed been provided in mice and humans (reviewed in Refs. 50 and 51). Available data suggest limited responses to most but not all (52) TLR ligands. Limitations in the capacity to release IL-12/IFN-g persist during the first year of life (53), and deficiencies in the numbers of APC and their functional competence limit the capacity to express effector memory responses (54). Future studies are expected to better define the relative influence of the immaturity of neonatal APC, neonatal T cells, and/or of the microenvironment in which APC/T-cell interactions take place, to indicate potentially effective immunomodulation strategies.

Influence of Maternal Antibodies on Neonatal CD4þ and CD8þ Vaccine Responses In contrast to the inhibiting influence of MatAb on infant antibody responses, MatAb may leave CD4þ and CD8þ T cell responses largely unaffected. This was first observed in mice, under experimental conditions in which high titers of MatAb completely abrogated antibody responses but did not affect either CD4þ T cell proliferative and cytokine responses (31,32) nor CTL responses (32,55,56). In human infants with MatAb who are immunized with measles and mumps vaccines, CD4þ T cell proliferative and INF-g responses remain unaffected, whereas antibody responses are inhibited (11,13,40). Accordingly, measles-specific T-cell responses were recorded in 86.8% of six-month old infants immunized in the presence of MatAb, whereas antibody responses were observed in only 36.7% (57). This inhibition of B-cell but not T-cell responses is best explained by the efficient uptake of antigen-antibody immune complexes by APC. Following processing, vaccine-derived antigenic peptides are thus presented at the APC cell surface, allowing priming of CD4þ and CD8þ T cells to occur independently of the inhibition of B cell responses. This early T-cell priming is likely to explain the reduced measles morbidity and mortality observed in vaccinated infants who failed to seroconvert due to the presence of maternal antibodies. It could also significantly facilitate early prime–later boost strategies in early human life, as shown in mice (32). To note, measles immunization of newborn macaques inhibited both B- and T-cell responses, suggesting that very high maternal antibody titers may totally prevent viral replication and subsequent responses (58).

Perspectives for Enhancement of Early-Life CD4þ and CD8þ Vaccine Responses Studies in mice have clearly demonstrated that adultlike T-cell responses may be induced even in the neonatal period if novel delivery systems and/or adjuvants are employed. This has been repeatedly achieved by DNA immunization against a panel of vaccine antigens (reviewed in Ref. 34), and the induction of adultlike CD4þ and CD8þ T-cell responses appears as a generic property of most DNA vaccines. In part, this could result from prolonged antigenic exposure, allowing both prolonged immune stimulation and ongoing immune maturation to occur. However, induction of adultlike neonatal Th1 and

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CTL responses were also achieved by certain adjuvants, including by oligonucleotides containing immunostimulating CpGmotifs (59,60), which are present in DNA plasmids, as well as by certain nonpersistent novel delivery systems. The current understanding is that neonatal T cells may have greater requirements than adult T cells for costimulatory signals, such that the induction of Th1 and CTL neonatal responses essentially reflects the relative capacity of vaccines to activate neonatal APCs to thresholds sufficient, or not, for optimal T-cell activation to occur. In mice, mimicking (IL-12 supplementation) or triggering (CD40) optimal APC activation is sufficient to induce adultlike IFN-g and CTL neonatal responses. Recently, the novel MF-59 adjuvant was reported as increasing human lymphoproliferative responses to recombinant HIV gp120 following immunization at birth, two weeks, two months, and five months of age (61), and ongoing studies with adjuvanted influenza vaccines are promising. It thus seems reasonable to expect that certain adjuvant formulations or delivery systems may prove capable of significantly enhancing early-life Th1/Th17 responses, representing a major progress in the control of early infections with intracellular pathogens. That these formulations may not be selected based on adult studies represents a significant challenge calling for specific early-life studies.

CONCLUSION AND PERSPECTIVES The rapid induction of strong and sustained antibody responses in very early life is yet an unmet challenge calling for a better understanding of the determinants of these important limitations. However, immune immaturity may not prevent early induction of memory B cells, which may be recalled by subsequent boosting. The limited capacity for early-life INFg and CTL responses appears to result essentially from suboptimal APC/T-cell interactions and thus might be overcome by use of specific adjuvants or delivery systems enhancing such interactions. As the optimal immunogenicity/reactogenicity balance of these new vaccine formulations will have to be defined in very young populations, attention must be directed to address the specific ethical and regulatory considerations of carrying out clinical trials in this age group.

ACKNOWLEDGMENTS The author is most grateful to the support provided by the Fondation Me´rieux and to all who participated in or supported the studies performed in the WHO Collaborating Center for Neonatal Vaccinology.

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26. Goldblatt D, Vaz AR, Miller E. Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate vaccines following infant immunization. J Infect Dis 1998; 177:1112–1115. 27. Belloni C, De Silvestri A, Tinelli C, et al. Immunogenicity of a three-component acellular pertussis vaccine administered at birth. Pediatrics 2003; 111:1042–1045. 28. Knuf M, Schmitt HJ, Wolter J, et al. Neonatal vaccination with an acellular pertussis vaccine accelerates the acquisition of pertussis antibodies in infants. J Pediatr 2008; 152:655–660, 660 e651. 29. Halasa NB, O’Shea A, Shi JR, et al. Poor immune responses to a birth dose of diphtheria, tetanus, and acellular pertussis vaccine. J Pediatr 2008; 153:327–332. 30. Markowitz LE, Albrecht P, Rhodes P, et al. Changing levels of measles antibody titers in women and children in the United States: impact on response to vaccination. Kaiser Permanente Measles Vaccine Trial Team. Pediatrics 1996; 97:53–58. 31. Siegrist CA, Cordova M, Brandt C, et al. Determinants of infant responses to vaccines in presence of maternal antibodies. Vaccine 1998; 16:1409–1414. 32. Siegrist CA, Barrios C, Martinez X, et al. Influence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime-boost strategies in mice. Eur J Immunol 1998; 28:4138–4148. 33. Ota MO, Vekemans J, Schlegel-Haueter SE, et al. Influence of Mycobacterium bovis bacillus Calmette-Guerin on antibody and cytokine responses to human neonatal vaccination. J Immunol 2002; 168:919–925. 34. Bot A. DNA vaccination and the immune responsiveness of neonates. Int Rev Immunol 2000; 19:221–245. 35. Prince AM, Whalen R, Brotman B. Successful nucleic acid based immunization of newborn chimpanzees against hepatitis B virus. Vaccine 1997; 15:916–919. 36. Bot A, Shearer M, Bot S, et al. Induction of antibody response by DNA immunization of newborn baboons against influenza virus. Viral Immunol 1999; 12:91–96. 37. Zaman K, Roy E, Arifeen SE, et al. Effectiveness of Maternal Influenza Immunization in Mothers and Infants. N Engl J Med 2008; 359(15):1555–1564. 38. Vekemans J, Amedei A, Ota MO, et al. Neonatal bacillus CalmetteGuerin vaccination induces adult-like IFN-gamma production by CD4þ T lymphocytes. Eur J Immunol 2001; 31:1531–1535. 39. Vekemans J, Ota MO, Wang EC, et al. T cell responses to vaccines in infants: defective IFNgamma production after oral polio vaccination. Clin Exp Immunol 2002; 127:495–498. 40. Gans HA, Maldonado Y, Yasukawa LL, et al. IL-12, IFN-gamma, and T cell proliferation to measles in immunized infants. J Immunol 1999; 162:5569–5575. 41. Gans HA, Yasukawa LL, Zhang CZ, et al. Effects of interleukin-12 and interleukin-15 on measles-specific T-cell responses in vaccinated infants. Viral Immunol 2008; 21:163–172. 42. Soares AP, Scriba TJ, Joseph S, et al. Bacillus Calmette-Guerin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles. J Immunol 2008; 180:3569–3577. 43. Miles DJ, van der Sande M, Crozier S, et al. Effects of antenatal and postnatal environments on CD4 T-cell responses to Mycobacterium bovis BCG in healthy infants in the Gambia. Clin Vaccine Immunol 2008; 15:995–1002. 44. Chiba Y, Higashidate Y, Suga K, et al. Development of cellmediated cytotoxic immunity to respiratory syncytial virus in human infants following naturally acquired infection. J Med Virol 1989; 28:133–139. 45. Mbawuike IN, Piedra PA, Cate TR, et al. Cytotoxic T lymphocyte responses of infants after natural infection or immunization with live cold-recombinant or inactivated influenza A virus vaccine. J Med Virol 1996; 50:105–111. 46. Jaye A, Magnusen AF, Sadiq AD, et al. Ex vivo analysis of cytotoxic T lymphocytes to measles antigens during infection and after vaccination in Gambian children. J Clin Invest 1998; 102:1969–1977.

Chapter 22: The Challenge of Vaccine Protection in Very Young Infants 47. Murray RA, Mansoor N, Harbacheuski R, et al. Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8þ T cell response. J Immunol 2006; 177:5647–5651. 48. He XS, Holmes TH, Mahmood K, et al. Phenotypic changes in influenza-specific CD8þ T cells after immunization of children and adults with influenza vaccines. J Infect Dis 2008; 197:803–811. 49. Adkins B. Development of neonatal Th1/Th2 function. Int Rev Immunol 2000; 19:157–171. 50. Velilla PA, Rugeles MT, Chougnet CA. Defective antigen-presenting cell function in human neonates. Clin Immunol 2006; 121:251–259. 51. Marchant A, Goldman M. T cell-mediated immune responses in human newborns: ready to learn? Clin Exp Immunol 2005; 141: 10–18. 52. Levy O, Suter EE, Miller RL, et al. Unique efficacy of Toll-like receptor 8 agonists in activating human neonatal antigen-presenting cells. Blood 2006; 108:1284–1290. 53. Chougnet C, Kovacs A, Baker R, et al. Influence of human immunodeficiency virus-infected maternal environment on development of infant interleukin-12 production. J Infect Dis 2000; 181:1590–1597. 54. Upham JW, Rate A, Rowe J, et al. Dendritic cell immaturity during infancy restricts the capacity to express vaccine-specific T-cell memory. Infect Immun 2006; 74:1106–1112.

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55. Martinez X, Li X, Kovarik J, et al. Combining DNA and protein vaccines for early life immunization against respiratory syncytial virus in mice. Eur J Immunol 1999; 29:3390–3400. 56. Bangham CR. Passively acquired antibodies to respiratory syncytial virus impair the secondary cytotoxic T-cell response in the neonatal mouse. Immunology 1986; 59:37–41. 57. Pabst HF, Spady DW, Carson MM, et al. Cell-mediated and antibody immune responses to AIK-C and Connaught monovalent measles vaccine given to 6 month old infants. Vaccine 1999; 17:1910–1918. 58. Premenko-Lanier M, Hodge G, Rota P, et al. Maternal antibody inhibits both cellular and humoral immunity in response to measles vaccination at birth. Virology 2006; 350:429–432. 59. Kovarik J, Bozzotti P, Love-Homan L, et al. CpG oligodeoxynucleotides can circumvent the Th2 polarization of neonatal responses to vaccines but may fail to fully redirect Th2 responses established by neonatal priming. J Immunol 1999; 162:1611–1617. 60. Brazolot Millan CL, Weeratna R, Krieg AM, et al. CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proc Natl Acad Sci U S A 1998; 95:15553–15558. 61. Borkowsky W, Wara D, Fenton T, et al. Lymphoproliferative responses to recombinant HIV-1 envelope antigens in neonates and infants receiving gp120 vaccines. AIDS Clinical Trial Group 230 Collaborators. J Infect Dis 2000; 181:890–896.

23 The Challenge of Inducing Vaccine Protection in the Elderly Dietmar Herndler-Brandstetter, Angelika Schwanninger, and Beatrix Grubeck-Loebenstein Immunology Division, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria

INTRODUCTION

VACCINE EFFICACY IN THE ELDERLY

In the last century, the morbidity and mortality caused by infectious diseases was dramatically reduced in developed countries because of improved standards of living and public health measures together with medical advances such as antibiotics and immunizations. This led to an unprecedented growth of the human population and to a rapid increase in average life expectancy, for instance from 49.2 years (1900) to 75.3 years (2000) in the United States. However, at the beginning of this century the demographic evolution in many developed countries arrived at a turning point, leading to population stagnation, because of low birth rates, and rapid aging (Fig. 1). This change in the population’s age structure, with an increase of the population aged 65 years and over from 21% in 2000 to 30% in 2050 (European Union), will pose an enormous medical and socioeconomic challenge on our future society. Aging is a multifactorial process characterized by the loss of function at the molecular, cellular, and organism level. A wide range of age-related alterations in immune system function have been described and are collectively referred to as immunosenescence. Clinically relevant is the higher prevalence, the more severe disease course, and the poorer prognosis of certain infectious diseases in the elderly population and the low efficacy of vaccinations. But also the development and progression of other age-related diseases, such as certain cancers, atherosclerosis, dementia, osteoporosis, and rheumatoid arthritis have been associated with impaired immune function in old age (1,2). Infectious diseases with a higher prevalence in elderly persons include respiratory tract infections (influenza and pneumonia are ranked among the ten major causes of deaths in the United States in persons aged 65 years and older), urinary tract infections, skin and soft tissue infections, Herpes zoster, tuberculosis, pneumococcal meningitis, and viral gastroenteritis (3). Furthermore, the increased global mobility of elderly persons may also enhance their risk of encountering newly emerging and reemerging infectious diseases. Although much progress has been achieved over the past decades in understanding the age-related changes of the immune system, the exact molecular mechanisms are not completely understood yet. Moreover, the development of vaccines that demonstrate a more favorable efficacy profile in elderly persons is still in its infancy. The goal of this chapter is to provide a comprehensive overview about age-related changes of the immune system influencing the outcome of vaccinations and will outline strategies to improve vaccine efficacy in elderly persons.

The implementation of large-scale vaccination strategies led to the eradication of smallpox in 1980 and to a drastic reduction of poliomyelitis, diphtheria, tetanus, pertussis, measles, mumps, rubella, and Haemophilus influenzae infections. More than 25 different infectious diseases can be prevented by vaccinations these days and vaccinations are considered the most cost-effective medical procedure for preventing morbidity and mortality caused by infectious diseases. However, the problem of decreased vaccine efficacy in the elderly, due to an age-related decline in immune system functions, has been recognized only recently. Especially the induction of protective antibody levels as well as functional and long-lived memory T-cell numbers in the case of vaccinations with new antigens remain a major problem in old age (Fig. 2). The situation of elderly persons is further aggravated by a different clinical presentation of infectious diseases, the failure to respond sufficiently to therapy, the frequent occurrence of opportunistic or recurrent infections and the reactivation of latent diseases, for instance those caused by varicella-zoster virus (VZV) and Mycobacterium tuberculosis.

Community-Acquired Infections Influenza is a contagious respiratory illness caused by the influenza virus strains A and B and is ranked among the 10 major causes of deaths among persons older than 65 years in the United States and other developed countries. Especially elderly persons and those with chronic conditions or otherwise immunocompromised persons have an increased risk of serious complications and death (4). The recurrent influenza epidemics are the consequence of point mutations (antigenic drift) of the viral surface proteins hemagglutinin and neuraminidase wherefore influenza vaccines need to be modified and applied annually. Although influenza vaccination coverage among elderly persons (65 years) increased from 15% to 20% (1980) to 65% (2001) in the United States, vaccine acceptance is lower in many other developed countries. Despite the availability of different types of influenza vaccines, the seroconversion after influenza vaccination is still disappointingly low among elderly persons (50% in persons aged 60 to 70 years, 31% for those aged 70 to 80 years, and only 11% for those above the age of 80; Table 1) (5), although reduced rates of hospitalizations and deaths have been attributed to influenza vaccination (6). The reduced vaccine efficacy correlates with lower levels of immunoglobulin A (IgA) and IgG antibodies, delayed antibody titers, and shortened maintenance of titers after vaccination.

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Figure 1 Probabilistic age pyramid of the European Union (25 members) for 2030. The grey line refers to the demographic situation in 2004. The black line predicts the EU-25 population age structure in 2030, including fractiles of the uncertainty distributions for the full age pyramid. The largest amount of uncertainty involves the ages below 10, while the graphic clearly demonstrates the increase of the population aged 60 years and above. Source: Courtesy of Vienna Institute of Demography, Data Sheet 2006.

Infection with Streptococcus pneumoniae is an important cause of invasive clinical manifestations, such as pneumonia, meningitis, and septicemia, and is the most common bacterial cause of community-acquired pneumonia in the elderly in developed countries. In the United States, the incidence of pneumococcal bacteremia and meningitis as well as associated mortality is highest among persons older than 65 years. An existing primary disease such as diabetes mellitus, liver disease, or chronic cardiovascular disease may even increase lethality up to 30%. The high mortality rates, in part, due to the increased occurrence of multiple drug-resistant pneumococcal strains are in favor of the current pneumococcal vaccine recommendations for all persons older than 65 years. Polysaccharide pneumococcal vaccines offer protection against invasive pneumococcal disease in 65% of the general elderly population, whereas it has only a moderate effect in high-risk groups. However, the still unsatisfying vaccine efficacy and the high costs of the vaccine are responsible for the low vaccination coverage among the elderly.

Infection with the VZV, an alphaherpesvirus, causes chickenpox, which is usually a mild disease in childhood. Thereafter, VZV remains latent in the dorsal root or cranial sensory ganglia until its reactivation because of decreased cell-mediated immunity. Hence, the incidence of Herpes zoster is high in persons older than 50 years, persons suffering from chronic diseases or receiving immunosuppressive therapy. The clinical manifestations are characterized by painful shingles and postherpetic neuralgia. Early initiation of antiviral therapy can reduce the severity of Herpes zoster, but it does not prevent the development of postherpetic neuralgia. Since 2006, a live-attenuated VZV vaccine has been on the market, which reduces the burden of illness due to Herpes zoster disease by 61.1% and the incidence of its most common sequela, postherpetic neuralgia, by 66.5% in adults aged 60 years or older (7). However, vaccine efficacy among persons older than 80 years is only around 18%. About 8 million people are infected worldwide by M. tuberculosis and most infected persons develop a latent infection, which can be reactivated in approximately 50% of the

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Vaccinations against tetanus, diphtheria, poliomyelitis, and pertussis have been administered on a routine basis since decades and have led to a significant reduction of morbidity and mortality. However, antibody levels after vaccination against tetanus, diphtheria, and pertussis decrease more rapidly in elderly persons (8,9). In contrast, longer-lasting protection and good responsiveness to boosting in spite of low antibody titers to poliomyelitis can be expected following exposure to live vaccine earlier in life (9).

Travel Vaccines

Figure 2 Antibody titers (Vienna Units/milliliter) against tick-borne encephalitis in young (<35 years; n ¼ 300) and elderly persons (>60 years; n ¼ 300) dependent on the time point of the last vaccination. In contrast to young persons, elderly persons had already significant lower antibody concentrations after one year. Antibody titers of >100 VIE U/mL are considered to be protective.

patients during their life, especially in old age when immunological competence declines. Tuberculosis is frequently diagnosed with delay in elderly persons because of an atypical clinical manifestation, which leads to enhanced morbidity and mortality. Further difficulties include the increased emergence of multiple drug-resistant strains with higher transmissibility and the poor efficacy of the current vaccine Bacillus Calmette-Gue´rin (BCG) in preventing the establishment of latent tuberculosis or reactivation of pulmonary disease in adults and the elderly. The BCG vaccine is therefore not recommended in many countries with a low incidence of tuberculosis. However, new tuberculosis vaccines have entered clinical trials and may also induce a strong cellmediated immune response, which is essential to protect from an intracellular pathogen such as M. tuberculosis.

Because of the increased mobility of the elderly, 5% to 8% of travelers in tropical areas are of advanced age, and this number is still increasing. Thus, the efficacy of travel vaccines protecting from typhoid and yellow fever, hepatitis A and B, tickborne encephalitis (TBE), Japanese encephalitis, and rabies is of increasing importance for the elderly who are dependent on a limited T- and B-cell repertoire that does not guarantee full responsiveness to new antigens. Nevertheless, in vitro experiments have demonstrated that T cells from elderly persons can still be stimulated by neoantigens, at least to the recombinant Etr protein of TBE virus and rabies virus (10). Hepatitis A is an acute disease of the liver caused by a nonenveloped virus belonging to the Picornaviridae family, with an estimated 1.5 million new infections per year worldwide. Hepatitis A vaccination is routinely recommended when traveling to tropic and subtropic areas. Clinical illness after hepatitis A infection is usually mild in young individuals, but the risk of severe infection and mortality increases with age. After combined hepatitis A/B vaccination, seroprotection was 92% in young adults (<40 years) compared with 63% for elderly persons (>60 years). It is therefore recommended to assess antibody levels in the elderly, as boosters have shown to be efficient in the case of vaccination failure (11). Another travel-related disease is yellow fewer, which is endemic in Africa and South America. Older adults possess an increased risk of severe disease, and mortality rates are highest in this age group. Because of the increased use of yellow fever vaccine in elderly persons, advanced age turned out to be a potential risk factor for severe adverse effects and even death. For elderly travelers, the risk for severe illness and death caused by yellow fever infection should therefore be carefully balanced against the risk for systemic illness due to the yellow fever vaccine (12). TBE is one of the most dangerous neuroinfectious diseases in Europe and Asia and is responsible for up to 12,000 cases of

Table 1 Vaccines Recommended for Routine Use in the Elderly Disease

Available vaccine types

Vaccine efficacya

Recommended booster intervals

Influenza

Split protein, subunit, virosome

<50%

Annually

Pneumonia

Conjugated polysaccharide

<65%

5–6 years

Tetanus Diphtheria Pertussis

Toxoid Toxoid Accellular

>84%b

5–10 years

Poliomyelitis

Inactivated virus

>99%c

10 years

Live-attenuated virus

<61%

ND

d

Herpes zoster

ND, not determined. a Overall vaccine efficacy in persons older than 60 years. b Vaccine efficacy after booster vaccination against tetanus, diphtheria and pertussis. c Vaccine efficacy after booster vaccination with an inactivated poliomyelitis virus, while for priming a live-attenuated vaccine was used. d Considered for future routine use in elderly persons.

Chapter 23: The Challenge of Inducing Vaccine Protection in the Elderly

TBE per year. Up to 30% of adults infected by the zoonotic arbovirus develop meningitis or meningoencephalitis and the lethality of TBE in Europe is up to 1%. The implementation of TBE vaccination with inactivated whole virus vaccines led to a dramatic decline of clinical cases. However, immunological responsiveness to TBE booster vaccination is dependent on the time of the last vaccination as well as age. Three to four years after TBE vaccination, 30% of people aged 60 years or older did not have protective TBE antibody levels, whereas 99% of people below the age of 40 were protected by the TBE vaccine (Fig. 2) (13). This emphasizes the need to follow regular booster vaccinations in old age to maintain protective antibody levels.

HOW DOES IMMUNOSENESCENCE INFLUENCE VACCINE EFFICACY? The aging process affects a wide range of cell types, including hematopoietic stem cells (HSCs), lymphoid progenitors, thymic epithelial cells, mature lymphocytes, as well as cells of the innate immune system. These age-related changes contribute to the decreased vaccine efficacy observed in the elderly in several ways. HSCs reside in the bone marrow, can give rise to all blood cell types including the myeloid and lymphoid lineages, and are long lived due to an extensive self-renewal capacity. However, studies on humans and animals indicate that HSCs show signs of aging. HSCs from elderly persons have a reduced capacity to differentiate into the lymphoid lineage, an increased expression of the cell-cycle inhibitor p16INK4A, and a decreased homing and reconstitution potential (14,15) (Fig. 3). Apart from these intrinsic defects, the age-related decline in hematopoietic

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tissue and an altered hematopoietic microenvironment may further contribute to defects in T and B cell progenitor cells and to a decline in lymphopoiesis during aging. The most prominent event during aging, however, is the continuous loss of thymic epithelial space beginning at the age of one year and resulting in a dramatic decline in thymopoiesis in old age (Fig. 4). The thymus, the central lymphoid organ, is responsible for the maturation and selection of antigeninexperienced, naive T cells that regenerate the peripheral T-cell pool and retain the capability of the adaptive immune system to respond to a variety of different pathogens. Agerelated changes in bone marrow stromal cells have also been shown to affect B-cell development, characterized by lower numbers of pre–B cells and fewer mature B cells that leave the bone marrow.

T Lymphocytes Despite the age-related decrease in thymopoiesis and the continuous exposure of the immune system to replicative stress through recurrent infections, the size of the peripheral T-cell pool remains stable throughout life. However, the composition of the peripheral T-cell pool changes during aging, with a dramatic decline in naive T-cell numbers and a concomitant increase of antigen-experienced T cells (Fig. 5). Although already low in numbers, peripheral naive T cells exhibit functional deficits in old age. These functional deficits comprise shortened telomeres, a restricted T-cell receptor (TCR) repertoire, impaired TCR-signaling, low interleukin (IL)-2 production, and an impaired generation of functional memory T cells (17). The functional impairments of peripheral naive T cells in

Figure 3 Age-associated alterations in lymphopoiesis. Pluripotent stem cells from the bone marrow give rise to lymphoid progenitors that differentiate into mature B and T lymphocytes in the bone marrow and the thymus, respectively. The most relevant age-related defects in T and B cell development are highlighted. See text for details.

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Figure 4 Age-dependent involution of the thymus. During aging, thymic cortex (black) and medulla (grey) are reduced in size, while adipose tissue (white) dominates the thymus in old age. Consequently, thymic output of recent thymic emigrants is decreased from >109 (1 year) to approximately 1.8  108 (>50 years). Source: From Ref. 16.

old age may originate from multiple factors, such as an increased post-thymic life span, homeostatic turnover, and/or long-term exposure to harmful environmental factors, which may affect not only the cytotoxic CD8 þ T-cell-mediated

response to neoantigens in old age but also B-cell-mediated responses that rely on adequate stimulation by CD4þ T-helper cells. While naive T-cell numbers decline with age, the life-long encounter of pathogens leads to the accumulation of antigenexperienced T cells. However, in old age, antigen-experienced T cells frequently display phenotypic as well as functional changes, which especially affect CD8þ T cells (18). The loss of the costimulatory molecule CD28 is one of the most consistent biological indicators of aging of the human immune system. CD28 T cells are long-lived lymphocytes with short telomeres, an increased resistance to apoptosis and a highly restricted TCR repertoire. The loss of CD28 is accompanied by the loss of another costimulatory molecule, CD27 and by the decreased expression of lymph node homing markers, L-selectin (CD62L), and chemokine receptor 7 (CCR7). The loss of these molecules leads to a decreased stimulation of T cells by antigenpresenting cells (APCs) and to an impaired migration into lymph nodes. In addition, changes in the membrane lipid composition of T lymphocytes during aging lead to an impaired formation of the immunological synapse, which further contributes to decreased T-cell activation and signaling (19). The differentiation into CD28 T cells is accompanied by the secretion of the type 1 cytokine interferon (IFN)-g and the loss of IL-2 production. The accumulation of terminally differentiated CD28 T cells thus contributes at least partly to the increased proinflammatory activity observed in the majority of elderly persons, while low CD28 T-cell numbers in old age

Figure 5 Age-related changes within the peripheral T-cell pool. The number of naive T cells decreases during aging, while antigenexperienced memory and effector T cells increase. In addition, peripheral naive as well as memory/effector T cells exhibit functional deficits in old age.

Chapter 23: The Challenge of Inducing Vaccine Protection in the Elderly

correlate with high IL-2 and IL-4 production, and good responsiveness to vaccinations (20,21). Furthermore, CD28 CD4þ T cells display decreased expression of CD154 (CD40L), which reduces their capacity to provide help for B-cell proliferation and antibody production. Although aging confers a major risk of morbidity and mortality from infectious diseases, chronic infections with cytomegalovirus (CMV), hepatitis C, human immunodeficiency virus (HIV), and Epstein-Barr virus may accelerate the aging process of the immune system and lead to premature immune senescence. Experimental evidence for this hypothesis comes from the identification of expanded, dysfunctional HIV- and CMV-specific CD28 T-cell clones in chronically infected persons (22,23). In longitudinal studies, CMV-seropositivity has been included in a set of immunological parameters (immune risk phenotype), which predict a twoyear mortality in the very elderly (24).

B Lymphocytes Similar to T cells, the number of peripheral B cells is maintained during aging, but each B-cell subset undergoes severe perturbations in size, dynamics, and repertoire. The changes that affect the B-cell subsets are due to a decreased generation of B-cell precursors, such as early lymphoid precursors and pro–B cells. Cell-intrinsic as well as microenvironmental disturbances are both likely to contribute to the decreased output of pro–B cells. Environmental factors also impair overall V(D)J recombinase activity among pro–B cells which, together with a decline in peripheral naive CD27þ B cells, accounts for the limited peripheral B-cell repertoire frequently detected in elderly persons (25). Of particular importance, impaired T-cell-mediated immunity as well as impaired stimulation by APCs also contribute to the decline in B-cell-specific function (26). For example, B cells from elderly individuals are stimulated 70% less efficiently by follicular dendritic cells (DCs) than B cells from young subjects. Additionally, germinal center reactions in lymph nodes, which are crucial for isotype switching and affinity maturation of antibodies, are impaired in old age because of the decline in CD4þ T-cell-mediated B-cell help. The molecular mechanisms of the dysregulated T-cell/B-cell interactions involve the loss of the costimulatory molecules CD27 and CD40L as well as the reduced production of IL-2 and IL-4 by CD28CD4þ T cells. As a consequence, primary antibody responses in elderly persons are frequently weak and short-lived and the produced antibodies bind with lower affinity (27).

Innate Immunity Innate immunity is dependent on a variety of cell types and mechanisms that provide the basis for an adequate response to pathogens. With increasing age, however, inflammatory processes occur ubiquitously and are referred to as ‘‘inflammaging’’ (28). This chronic inflammation can support the development and progression of age-related diseases, such as atherosclerosis, rheumatoid arthritis, osteoporosis, and neurodegeneration. The severity of chronic inflammatory processes in elderly people also depends on genetic factors, such as polymorphisms within genes encoding for cytokines like IL-6, IL-10, and IFN-g, and have been associated with changes in life span (29). Functional deficits of innate immune system components may lead to the inability to eliminate pathogens and may consequently trigger the chronic activation of nonspecific responses. For example, neutrophils produce reduced amounts of superoxide anion and exhibit changes in membrane fluidity

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and chemotaxis. Of great relevance for vaccine efficacy is the question of how aging affects professional APCs. The recognition and uptake of antigen initializes a maturation program within DCs and leads to the upregulation of major histocompatibility complex (MHC) and costimulatory molecules. Adequate stimulation of DCs is therefore a prerequisite for proper T-and B-cell responses. However, the impact of aging on DC function has not been fully elucidated yet. Experiments in mice suggest that the density of DCs in the skin, the expression of MHC class II and other cell-surface molecules, and the capacity of DCs to present antigen can all be altered with increasing age (30). Though, only few studies have been carried out to analyze these effects in humans (31).

STRATEGIES TO ENHANCE THE IMMUNOGENICITY OF VACCINES Because of the reduced protective effect of vaccinations and the high morbidity and mortality from infectious diseases in old age, there is a tremendous need to improve vaccine efficacy. Vaccines that target an old immune system need to stimulate CD4þ T-helper cells and B cells more efficiently to enhance antibody responses and ensure the formation of long-lasting memory. Furthermore, cell-mediated immunity has been shown to play a key role in protection from influenza, Herpes zoster, tuberculosis, typhoid fever, and hepatitis A and B (32). For these infectious diseases it is therefore of utmost importance to induce functional and long-lasting memory CD8þ T cells. Several strategies are being pursued to enhance the efficacy of vaccines and to minimize adverse side effects (33,34). Live-attenuated vaccines have been proven highly efficient in eliciting T- and B-cell-mediated immunity, while conjugate and subunit vaccines have a very favorable safety profile but need to be supplemented with adjuvants to enhance immunogenicity (35). Adjuvants can be classified into antigen delivery systems [e.g., aluminium salts, microparticles, liposomes, oil-in-water emulsions, and immunostimulatory complexes (ISCOMs)] and immune potentiators [the saponin component QS21, 3-deacetylated monophosphoryl lipid (MPL) A, oligodeoxynucleotides-containing CpG motifs (CpG-ODNs), cytokines and nucleic acids]. The mechanisms of action of these adjuvants are to improve antigen processing and presentation, and to stimulate innate immunity components. In particular, antigen delivery systems convert soluble antigens into particulate material, which is more readily ingested by APCs. In contrast, immune potentiators stimulate innate immune components through evolutionary conserved pathogen recognition receptors or modulate T- and B-cell responses through the application of DNA encoding for cytokines, costimulatory molecules, or chemokines. However, only few adjuvants are licensed for human use. Aluminium salts have been used widely in humans to enhance specific antibody responses but they have little capacity to stimulate cell-mediated immunity (36). Until now, only two other vaccine adjuvants have been approved for use in human influenza vaccines: an oilin-water emulsion (MF591), which is used as an adjuvant in subunit influenza vaccines, and a virosomal influenza vaccine. These adjuvanted vaccines demonstrate an improved immunogenicity in elderly persons with seroconversion rates up to 68% (37,38). Numerous other adjuvants are currently being tested in animal models and clinical trials. Immunostimulatory adjuvants may overcome the proposed age-related functional declines of innate and adaptive immune responses.

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In addition to improve vaccine efficacy, a modification of vaccination strategies for elderly persons has been supported by the results of several vaccination trials. A decreased response and a shortened duration of protective immunity following booster immunization is a characteristic feature of old age (13). In Austria, for example, health authorities have therefore recommended five-year vaccination intervals for tetanus, diphtheria, pertussis, and pneumonia. Increased public awareness of regular booster vaccinations in adults should be enforced, as these immunization regimes may be essential to maintain the ability to respond to recall antigens in old age. Recent results also indicate that not only long-lasting protection but also a good booster effect can be expected even a long time after the last vaccination, when a live-attenuated vaccine (e.g., polio vaccine) is used for primary immunization in early life (9). New delivery systems that make use of tiny microneedles or noninjectable application devices may further increase vaccination acceptance, especially in the case of influenza, as this vaccination has to be repeated annually. In the distant future, strategies to reverse or delay immunosenescence may also become apparent. Because thymic involution is a key event for the age-related deterioration of immune function, therapies aiming at promoting thymic regrowth, and increasing thymopoiesis are currently under investigation. The administration of IL-7 or growth hormone and insulin-like growth factor 1 have been proven promising in animal models. Furthermore, the eradication of chronic bacterial or viral infections may further delay immunosenescence. In animal models, a 30% reduction of caloric intake has also been found to slow multiple aspects of aging, such as the age-related loss of naive T cells and the decreased proliferative capacity after antigenic stimulation (39). Whether caloric restriction has similar effects in humans has not been proven yet.

CONCLUDING REMARKS By virtue of the demographic development taking place in developed countries, infectious diseases in elderly persons have gained increasing importance. Thus, the development of more immunogenic vaccines for the elderly is relevant to the protection of public health. The improvement of specific vaccine types regarding immunogenicity and tolerability, the addition of adjuvants, the design of new delivery systems as well as specific immunization regimes should all contribute to enhanced efficacy of vaccines in elderly persons. For the short term, improvements could be achieved by raising people’s awareness regarding recommended booster vaccination intervals throughout life and by adjusting vaccination intervals in old age. The enhanced efficacy of vaccines and the introduction of needle-free injection devices are likely to increase vaccine acceptance and vaccination coverage among elderly persons.

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5. Bouree P. Immunity and immunization in elderly. Pathol Biol (Paris) 2003; 51(10):581–585. 6. Nichol KL, Margolis KL, Wuorenma J, et al. The efficacy and cost effectiveness of vaccination against influenza among elderly persons living in the community. N Engl J Med 1994; 331(12): 778–784. 7. Oxman MN, Levin MJ, Johnson GR, et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med 2005; 352(22):2271–2284. 8. Grubeck-Loebenstein B, Berger P, Saurwein-Teissl M, et al. No immunity for the elderly. Nat Med 1998; 4(8):870. 9. Kaml M, Weiskirchner I, Keller M, et al. Booster vaccination in the elderly: their success depends on the vaccine type applied earlier in life as well as on pre-vaccination antibody titers. Vaccine 2006; 24(47–48):6808–6811. 10. Gomez I, Marx F, Gould EA, et al. T cells from elderly persons respond to neoantigenic stimulation with an unimpaired IL-2 production and an enhanced differentiation into effector cells. Exp Gerontol 2004; 39(4):597–605. 11. Wolters B, Junge U, Dziuba S, et al. Immunogenicity of combined hepatitis A and B vaccine in elderly persons. Vaccine 2003; 21(25–26): 3623–3628. 12. Martin M, Weld LH, Tsai TF, et al. Advanced age a risk factor for illness temporally associated with yellow fever vaccination. Emerg Infect Dis 2001; 7(6):945–951. 13. Hainz U, Jenewein B, Asch E, et al. Insufficient protection for healthy elderly adults by tetanus and TBE vaccines. Vaccine 2005; 23(25):3232–3235. 14. Rossi DJ, Bryder D, Zahn JM, et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A 2005; 102(26):9194–9199. 15. Janzen V, Forkert R, Fleming HE, et al. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 2006; 443(7110):421–426. 16. Haynes BF, Markert ML, Sempowski GD, et al. The role of the thymus in immune reconstitution in aging, bone marrow transplantation, and HIV-1 infection. Annu Rev Immunol 2000; 18: 529–560. 17. Haynes L, Eaton SM. The effect of age on the cognate function of CD4þ T cells. Immunol Rev 2005; 205:220–228. 18. Effros RB, Cai Z, Linton PJ. CD8 T cells and aging. Crit Rev Immunol 2003; 23(1–2):45–64. 19. Huber LA, Xu QB, Jurgens G, et al. Correlation of lymphocyte lipid composition membrane microviscosity and mitogen response in the aged. Eur J Immunol 1991; 21(11):2761–2765. 20. Saurwein-Teissl M, Lung TL, Marx F, et al. Lack of antibody production following immunization in old age: association with CD8(þ)CD28() T cell clonal expansions and an imbalance in the production of Th1 and Th2 cytokines. J Immunol 2002; 168(11): 5893–5899. 21. Schwaiger S, Wolf AM, Robatscher P, et al. IL-4-producing CD8þ T cells with a CD62Lþþ(bright) phenotype accumulate in a subgroup of older adults and are associated with the maintenance of intact humoral immunity in old age. J Immunol 2003; 170(1): 613–619. 22. Appay V, Dunbar PR, Callan M, et al. Memory CD8þ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002; 8(4):379–385. 23. Almanzar G, Schwaiger S, Jenewein B, et al. Long-term cytomegalovirus infection leads to significant changes in the composition of the CD8þ T-cell repertoire, which may be the basis for an imbalance in the cytokine production profile in elderly persons. J Virol 2005; 79(6):3675–3683. 24. Hadrup SR, Strindhall J, Kollgaard T, et al. Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly. J Immunol 2006; 176(4):2645–2653.

Chapter 23: The Challenge of Inducing Vaccine Protection in the Elderly 25. Weksler ME, Szabo P. The effect of age on the B-cell repertoire. J Clin Immunol 2000; 20(4):240–249. 26. McGlauchlen KS, Vogel LA. Ineffective humoral immunity in the elderly. Microbes Infect 2003; 5(13):1279–1284. 27. Johnson SA, Cambier JC. Ageing, autoimmunity and arthritis: senescence of the B cell compartment—implications for humoral immunity. Arthritis Res Ther 2004; 6(4):131–139. 28. Franceschi C, Bonafe M, Valensin S, et al. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci 2000; 908:244–254. 29. Pes GM, Lio D, Carru C, et al. Association between longevity and cytokine gene polymorphisms. A study in Sardinian centenarians. Aging Clin Exp Res 2004; 16(3):244–248. 30. Grewe M. Chronological ageing and photoageing of dendritic cells. Clin Exp Dermatol 2001; 26(7):608–612. 31. Plackett TP, Boehmer ED, Faunce DE, et al. Aging and innate immune cells. J Leukoc Biol 2004; 76(2):291–299. 32. Salerno-Goncalves R, Sztein MB. Cell-mediated immunity and the challenges for vaccine development. Trends Microbiol 2006; 14(12): 536–542.

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24 Vaccination and Autoimmunity Paul-Henri Lambert Centre of Vaccinology and Department of Pathology, University of Geneva, Geneva, Switzerland

Michel Goldman Institute for Medical Immunology, Universite´ Libre de Bruxelles, Charleroi, Belgium

INTRODUCTION Diseases encompassing manifestations caused by an autoimmune process are not infrequent and are known to appear in age groups that are often selected as targets for vaccination programs. Therefore, in the context of a rapidly increasing number of vaccination events, it may not be surprising that the question of a potential interaction between vaccines and autoimmune diseases is being raised with an increasing insistence. It is estimated that as much as 5% of the population in Western countries suffers from autoimmune diseases (1). These disorders represent a growing burden for health budgets as their incidence has significantly increased over the past years, as documented for type I diabetes (2) and multiple sclerosis (MS) (3). It is generally assumed that autoimmune disorders result from complex interactions between genetic traits and environmental factors. Indeed, although there is a frequent concordance of autoimmune diseases among monozygotic twins (4), the concordance rate is lower than expected. Similarly, changes in the incidence of type I diabetes and MS when children from a given population migrate from one region to another (5,6) strongly suggest a critical role for environmental causes in addition to genetic predisposition. In most autoimmune diseases, the trigger has not been formally identified, leaving room for hypotheses and allegations not always substantiated by facts. Mechanisms leading to autoimmune responses and their occasional translation into autoimmune diseases are now better understood. Autoimmune responses result from the combined effects of antigen-specific stimulations of the immune system and an antigen nonspecific activation of antigen-presenting cells in the context of a genetically determined predisposition. Most often, such responses are not followed by any clinical manifestations unless additional events favor disease expression, for example, a localized inflammatory process at tissue level. Infections have occasionally been demonstrated either as etiologic factors or as triggering events in autoimmune diseases. Well-known examples are post-streptococcal (Group A Streptococcus pyogenes) heart disease or the Guillain-Barre´ syndrome (GBS) that follows Campylobacter jejuni infections. Such observations have emphasized the multifactorial immunological pathogenesis of secondary autoimmune pathology. First, there is a potential role of antigenic similarities between some microbial molecules and host antigens (antigenic mimicry). Second, infection-related signals that trigger innate immunity

appear to play an essential role in enhancing the immunogenicity of host antigens or of host-mimicking epitopes, and in possibly overcoming regulatory mechanisms that limit autoimmune responses. It should be stressed that post-infectious autoimmune responses are not infrequent whereas associated autoimmune diseases remain rare events and often require additional infection-related inflammatory processes. It is on the basis of such observations that questions are raised regarding the potential risk of autoimmune responses and of autoimmune diseases following vaccination. Is there a significant risk that some vaccines may induce autoimmune responses through the introduction of microbial epitopes that cross-react with host antigens? Can adjuvant-containing vaccines trigger the clinical expression of an underlying autoimmune process through a ‘‘nonspecific’’ activation of antigenpresenting cells and the release of inflammatory cytokines? Until now, answers to these questions have been largely based on epidemiological studies, with limitations due to the difficulty to assess the frequency of relatively rare events during clinical trials or post-marketing surveillance. When considered as a whole, autoimmune diseases affect up to 3% of the general population in industrialized countries, but many specific autoimmune diseases have a relatively low natural incidence. Whereas diseases such as rheumatoid arthritis may reach 1% prevalence, others such as MS or systemic lupus erythematosus (SLE) are less frequent (around 0.1%) and many others are relatively rare diseases. Therefore, for most of these clinical entities, only very large epidemiological studies or huge clinical trials may allow for a consistent assessment of the relative risk of vaccine-related effects. Understanding the mechanisms by which autoimmune responses are generated and how they may or may not lead to autoimmune diseases is of paramount importance for defining the real risk of vaccine-associated autoimmune reaction. During the course of vaccine development, it is now becoming conceivable that a comprehensive and multidisciplinary approach would help to reduce to a minimum the risk that a new vaccine would induce autoimmune manifestations. Later, once the new vaccine is largely used in public health programs, systems should be in place to readily assess observations or allegations of unexpected autoimmune adverse effects. Although the past few years have seen a dramatic increase in the number of such allegations, it is somewhat reassuring that autoimmune adverse effects were demonstrated in only very few instances.

Chapter 24: Vaccination and Autoimmunity

INFECTIOUS AGENTS AS TRIGGER OF AUTOIMMUNE DISEASES The prototype of autoimmune disease of infectious origin is rheumatic fever. It is caused by an anti-streptococcal immune response that cross-reacts with cardiac myosin (7). Another welldocumented example is the GBS occurring in the course of Campylobacter jejuni infection and is mediated by anti-bacterial lipopolysaccharide antibodies that cross-react with human gangliosides (8). Similarly, antibodies directed against the Tax protein of the human T-lymphotropic virus type 1 (HTLV-1) and crossreacting with the heterogeneous nuclear ribonucleoprotein-A1 (hnRNP-A1) self-antigen were demonstrated in HTLV-1associated myelopathy/tropical spastic paraparesis (9). Although cross-reactivity between viral peptides and self-antigens was documented in type I diabetes and MS and despite circumstantial observations linking the overexpression of these diseases to previous viral infections, a clear-cut relation between the onset of organ-specific autoimmunity and viral infection has not been firmly established except for type 1 diabetes in the context of congenital rubella (10–12). The role of viruses is also suspected in systemic autoimmune diseases, especially SLE. However, such a role has only been clearly demonstrated in mixed cryoglobulinemia, a disease associated with hepatitis C (13). It has also been proposed that some long-term complications of infections might be of autoimmune origin. This is the case for reactive arthritis consecutive to infection with intracellular bacteria, including Chlamydia, Salmonella, Shigella, Borrelia, and Yersinia spp. In these diseases, there is evidence of a persistent pathogenic immune response involving T lymphocytes, but whether such T-cell responses are directed against cross-reactive self-antigens or maintained by persistent bacterial antigens is still an open question (14,15). In Lyme arthritis, the identification of an immunodominant epitope of the outer surface protein A of Borrelia burgdorferi (Osp A) displaying significant homology with human LFA-1, an adhesion molecule of the b2 integrin family, provided convincing evidence for an autoimmune mechanism (16). Indeed, cross-reactive T-cell responses to OspA and LFA-1 were observed in blood and synovial fluid of patients with antibiotic-resistant chronic Lyme arthritis (16). The role of infections as etiological agents of human autoimmune disease has been demonstrated in only few instances. However, their involvement in the exacerbation of a preexisting autoimmune disorder is rather well established. For example, in MS, epidemiological data strongly suggest that relapses of the disease can be triggered by both bacterial and viral infections (17,18). Several vaccine-preventable infections are well known to negatively influence the course of defined autoimmune diseases. Vaccination in such cases is highly recommended (e.g., influenza vaccination in patients with MS) (19) since no exacerbation has been recorded following the use of any of the current vaccines.

MECHANISMS OF AUTOIMMUNITY INDUCED BY INFECTIOUS AGENTS It is generally assumed that activation and clonal expansion of autoreactive T lymphocytes represent critical steps in the pathogenesis of autoimmune diseases. Infections might be responsible for these key events through several nonmutually exclusive mechanisms including molecular mimicry, enhanced presentation of self-antigens, bystander activation, and impaired T-cell regulation (15).

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Molecular Mimicry The molecular mimicry hypothesis is based on sequence homologies between microbial peptides and self-antigen epitopes. At the T-cell level, this concept was initially established in an experimental model in which immunization with a hepatitis B (HB) virus polymerase peptide containing a six amino acid sequence of rabbit myelin basic protein (MBP) elicited an anti-MBP T-cell response leading to autoimmune encephalomyelitis (EAE) (20). The demonstration that a viral infection in itself can lead to autoimmune pathology caused by molecular mimicry was established in a murine model of herpes simplex keratitis in which pathogenic autoreactive T-cell clones were shown to cross-react with a peptide from the UL6 protein of the herpes simplex virus (21). Indeed, a single amino acid mutation in the UL6 T-cell epitope was sufficient to limit the capacity of the mutant virus to induce autoimmune corneal lesions (22). Conclusive evidence that a viral infection can induce pathogenic autoreactive T cells was also provided in a model of Theiler’s murine encephalomyelitis virus encoding a mimicking peptide (23). Molecular mimicry at the level of epitopes recognized by CD8þ T lymphocytes may also be involved in autoimmunity. This was shown in a model of inflammatory bowel disease induced in immunodeficient mice by CD8þ T-cell clones directed against mycobacterial heat shock protein hsp60, which crossreact with hsp60 self-antigen (24). B-cell epitope mimicry also occurs. Functional mimicry of host proteins may be quite widespread, as it may allow pathogens not only to evade an immune response but also to use cellular receptors as port of entry. Such functional mimicry of human glycosphingolipids by lipopolysaccharides (LPS) from several Neisseria sp. and from Haemophilus influenzae may have evolved to serve this function (25). Structural homology with autoimmune implications has been identified. A tetrasaccharide of the LPS core of the gastrointestinal pathogen Campylobacter jejuni can induce antibodies to human gangliosides and may be causally implicated in the autoimmune GBS (26). It is also well known that Lewis-like polysaccharide antigens from certain Helicobacter pylori strains induce antibodies that cross-react with gastric mucosa antigens and appear to contribute to atrophic gastritis in man (27).

Enhanced Presentation of Self-Antigens Infection can promote processing and presentation of selfantigens by several mechanisms. First, cellular damages locally induced by viral or bacterial infection can result in the release of sequestered self-antigens that stimulate autoreactive T cells. This was clearly demonstrated in autoimmune diabetes induced by coxsackievirus B4 infection in mice (28). Second, the local inflammatory reaction elicited in tissues by microbial products can trigger dendritic cell maturation, which represents a key step in the induction phase of immune responses. Microbial products that engage toll-like receptors on dendritic cells can induce the upregulation of membrane expression of major histocompatibility complex (MHC) and costimulatory molecules and the secretion of cytokines, particularly interleukin (IL)-12, which promote T-cell activation (29). Third, a T-cell response directed toward a single self-peptide can ‘‘spread’’ to other self-epitopes during an inflammatory reaction. This process of ‘‘epitope spreading’’ has been well documented in murine models of encephalomyelitis (15).

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Bystander Activation The release of cytokines such as IL-12 can promote bystander activation of memory T cells and occasionally trigger autoimmune reactions when such autoreactive cells do preexist. Using murine models of encephalomyelitis, Shevach and co-workers demonstrated that quiescent autoreactive T cells could differentiate into pathogenic Th1 effectors in presence of microbial products that induce IL-12 synthesis (30,31). Likewise, Fujinami and co-workers demonstrated that IL-12 inducing viral infections could elicit relapses of EAE, in a nonantigen-specific manner, in myelin-primed animals (32). It now appears that the critical T-cell subset responsible for autoimmune pathology might be the Th17 subset and not the Th1 subset as previously assumed (33). Along this line, the most important IL-12 family member for the induction of autoimmune inflammation appears to be IL-23, which promotes TH17 activation rather than IL-12p70, which promotes Th1 activation (34). A salient feature of bystander activation is its limited duration. To observe an exacerbation of EAE, one should provide the triggering signal within a relatively restricted window of time after the etiological stimuli that ‘‘primed’’ the animal for disease. In addition, disease exacerbation occurs within weeks after bystander activation, and it is not usually seen after longer delays (32).

2. 3.

4.

Regulatory T Cells There is growing evidence that regulatory T cells are instrumental in controlling autoreactive T cells both in neonates and adults (35,36). Indeed, depletion of regulatory CD25þ T cells promotes autoimmunity, although in adult animals this maneuver is not sufficient by itself and requires administration of self-antigen (36). It is likely that infectious agents can have profound influence, either positive or negative, on regulatory T cells. Indeed, the balance between TH-17 and regulatory T cells is rather delicate (33). Vaccine adjuvants inducing high levels of IL-6 might indeed inhibit regulatory T-cell differentiation and activities while promoting TH-17 responses (33). On the other hand, there is recent evidence that TLR4 ligation might induce the emergence of regulatory T cells (37). This represents an increasingly important area of investigation that will probably deserve attention during the course of vaccine development.

An association between vaccine administration and an autoimmune adverse event is most likely to be considered strong when the evidence is based on

1.

THE RISK OF VACCINE-ASSOCIATED AUTOIMMUNITY

There exist no general criteria for diagnosing vaccine-related autoimmune disease, and this question has to be analyzed on a case-by-case basis. In general, appropriate epidemiological studies are essential before seriously considering that a particular autoimmune clinical condition might be associated with a given vaccination. This can then be followed by the determination of known biological markers of the identified autoimmune disease in other vaccinees. However, it is always relevant to compare the level of vaccine-related risk to that associated with the corresponding natural infection, for the population at large or for specific subgroups to be identified. Criteria underpinning vaccine adverse event causality assessment have been established by WHO (38). Some of these criteria particularly apply to autoimmune diseases and may be summarized as follows:

1.

Consistency. The association of a purported autoimmune event with the administration of a vaccine should be

consistent; that is, the findings should be replicable in different localities, by different investigators not unduly influencing one another, and by different methods of investigation, all leading to the same conclusion(s). Strength of the association. The association should be strong in the magnitude of the association (in an epidemiological sense). Specificity. The association should be distinctive and the adverse event should be linked uniquely or specifically with the vaccine concerned, rather than its occurring frequently, spontaneously or commonly in association with other external stimuli or conditions. An adverse event may be caused by a vaccine adjuvant or additive, rather than by the active component of the vaccine. In this case, it might spuriously influence the specificity of the association between vaccine and adverse event. Temporal relation. There should be a clear temporal relationship between the vaccine and the adverse event, in that receipt of the vaccine should precede the earliest manifestation of the event or a clear exacerbation of an ongoing condition. The timing is important; long delays (over 2 months) are not the rule. Indeed, the induction or the acceleration of autoimmune tissue lesions that have been observed following some acute infections (e.g., Campylobacter jejuni or influenza) has always occurred within weeks after the infectious event.

2.

3.

4.

Well-conducted human studies that demonstrate a clear association in a study design that is determined a priori for testing the hypothesis of such association. Such studies will normally be one of the following, in descending order of probability of achieving the objective of the study: randomized controlled clinical trials, cohort studies, case control studies, and controlled case-series analyses. Case reports, however numerous and complete, do not fulfill the requirements for testing hypotheses. When autoimmune events appear attributable to a vaccine, it is important to determine whether there is a predisposed set of subjects (by age, population, genetic, immunological, environmental, ethnic, sociological, or underlying disease conditions). Such predisposition is most likely to be identified in case-controlled studies. An association that is demonstrated in more than one human study and consistent among the studies. The studies would need to have been well conducted, by different investigators, in different populations, with results that are consistent, despite different study designs. In the case of future vaccines against infections known to be associated with autoimmune complications (e.g., postgroup A streptococcal rheumatic heart disease), vaccineassociated autoimmune adverse events that closely resemble these infection-associated complications. A nonrandom temporal relationship between administration and the adverse incident. There should be a strict definition of the autoimmune adverse event in clinical, pathological, and biochemical terms, as far as that is achievable. The frequency in the nonimmunized population of the adverse event should be substantially different from that in the immunized population.

Chapter 24: Vaccination and Autoimmunity

Vaccine-Attributable Autoimmune Diseases It is only in a few rare cases that autoimmune pathology has been firmly considered as attributable to the use of modern vaccines. For example, a form of GBS, polyradiculoneuritis, was found associated with the 1976 to 1977 vaccination campaign against swine influenza using the A/New Jersey/8/76 swineflu vaccine (39). The estimated attributable risk of vaccinerelated GBS in the adult population was just under one case per 100,000 vaccinations, and the period of increased risk was concentrated primarily within the five-week period after vaccination (relative risk: 7.60). Although this original Centers for Disease Control study demonstrated a statistical association and suggested a causal relation between the two events, controversy has persisted for several years. The causal relation was reassessed and confirmed in a later study focusing on cases observed in Michigan and Minnesota (40). The relative risk of developing GBS in the vaccinated population of these two states during the six weeks following vaccination was 7.10 whereas the excess cases of GBS during the first six weeks attributed to the vaccine was 8.6 per million vaccinees in Michigan and 9.7 per million vaccinees in Minnesota. The pathogenic mechanisms involved are still unknown. With subsequent influenza vaccines, no significant increase in the development of GBS was noted (41), and it is currently assumed that the risk of developing GBS following vaccination (one additional case per million persons vaccinated) is substantially less than the risk for severe influenza and influenzarelated complications (42). Another example of confirmed autoimmune adverse effect of vaccination is idiopathic thrombocytopenia (ITP) that may occur after measles-mumps-rubella (MMR) vaccination (43–47). The reported frequency of clinically apparent ITP after this vaccine is around 1 in 30,000 vaccinated children. In one study (43), the relative incidence in the six-week postimmunization risk period has been estimated to be 3.27 (95% CI, 1.49–7.16) when compared to the control period. In about two-thirds of the patients, platelet counts under 20,000 have been recorded. The clinical course of MMR-related ITP is usually transient but it is not infrequently associated with bleeding and, as shown in a study conducted in Finland, it can occasionally be severe (48). In this latter study, there was an increase in platelet-associated immunoglobulin in 10 of 15 patients, whereas circulating antiplatelet autoantibodies, specific for platelet glycoprotein IIb/IIIa, were detected in 5 of 15 patients. These findings are compatible with an autoimmune mechanism triggered by immune response to MMR vaccination. However, it should be noted that the risk for thrombocytopenia following natural rubella (1/3000) or measles (1/6000) infections is much greater than after vaccination (42). Patients with a history of previous immune thrombocytopenic purpura are prone to develop this complication, and in these individuals the risk of vaccination should be weighed against that of being exposed to the corresponding viral diseases (49).

Vaccine-Related Allegations of Autoimmune Adverse Effects The advent of new vaccines and the increasing number of highly publicized reports that claim a link between certain immunizations and autoimmune disease have led to public concern over the risk of inducing autoimmune disease by immunization. For example, special concerns have been voiced recently in France regarding the potential association of MS

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with HB vaccination. Similarly, questions have been raised in the United States whether childhood vaccinations influence the rate of occurrence of type 1 diabetes. Such allegations, even if they are not confirmed, may have detrimental effects on vaccination programs at a global level and therefore require particular attention. Hepatitis B and Multiple Sclerosis The possible association of HB vaccination with the development of MS was primarily questioned in France, following the report of 35 cases of primary demyelinating events occurring at one Paris hospital between 1991 and 1997, within eight weeks of recombinant HB vaccine injection (50–52). The neurological manifestations were similar to those observed in MS. There were inflammatory changes in the cerebrospinal fluid and high signal intensity lesions were observed in the cerebral white matter on T2-weighted MR images. After a mean follow-up of three years, half of them became clinically definite MS. These neurological manifestations occurred in individuals considered at higher risk for MS: a preponderance of women, mean age near 30 years, overrepresentation of the DR2 HLA antigen, and a positive family history of MS. These observations rapidly called the attention of the French pharmacovigilance system, and from 1993 through 1999, several hundred cases with similar demographic and clinical characteristics were identified. It is essential to note that this episode occurred in a very special context. In France, close to 25 million people received the HB vaccine during this period, of which 18 million were adults, and this represented about 40% of the total country population. No case was reported in children less than three years. Since these initial reports, at least 10 studies aiming at defining the significance of such observations have now been completed. They are summarized on Table 1. There was no significant association between HB vaccination and the occurrence of demyelinating events or MS in any of these studies. However, a common feature was an insufficient statistical power to definitely exclude such an association. Two studies are particularly illustrative of the difficulty of interpreting these data. First, a retrospective, hospital-based case-control study was carried out on patients experiencing the first episode of central nervous system (CNS) demyelination during the two–year period January 1994 to December 1995 (55) (121 cases and 121 matched controls). Adjusted odds ratio (OR) obtained from conditional logistic regression between a CNS demyelination and HB vaccine exposure during the previous 60 days, were 1.7 (95% CI, 0.5–6.3) and, during the previous 61 to 180 days, 1.5 (95% CI, 0.5–5.3). Second, a population-based case-control study using the general practice database in the United Kingdom analyzed 360 cases with incident MS and 140 cases of central demyelination. Each case was matched with up to six controls (63). The OR for exposure to HB vaccine in the 0 to 12 months period was 1.6 (95% CI, 0.6–4.0). However, two recent studies bear a particular weight in confirming the lack of a significant association between HB vaccination and the occurrence of MS (54). Confavreux et al. conducted a case-crossover study in patients included in the European Database for MS who had a relapse between 1993 and 1997. The index relapse was the first relapse confirmed by a visit to a neurologist and preceded by a relapse-free period of at least 12 months. Exposure to vaccination in the two-month risk period immediately preceding the relapse was compared with that in the four previous two-month control periods for the

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Table 1 Clinical Studies of the Association Between MS or Demyelinating Diseases with Hepatitis B Vaccination Analysis

Study site

RR/OR (time interval)

CI 95%

References

MS, 1st episode

USA Europe France

0.3–1.8 0.5–1.6 0.4–1.3 0.5–6.3 0.5–5.3

Ascherio et al., 2001 (53)

MS, relapses Acute demyelinating disease MS, 1st episode

Canada

MS, 1st episode

USA

Acute demyelinating disease MS, relapses

USA France

Acute demyelinating disease

France

MS, 1st episode & acute demyelinating disease Acute demyelinating disease

UK

0.7 (24 months) 0.9 (any time) 0.71 (2 months) 1.7 (2 months) 1.5 (2 to –6 months) 5/288,657 (prevaccination period, 1986–1992) 9/289,651(postvaccination period, 1992–1998) 1.3 (6 months) 1.0 (12 months) 2.0 0.9 (36 months) 1.09 0.6/yr (incidence before vaccination) 0.5/yr (incidence after vaccination) 1.05 (2 months, expected 102.7 vs. observed 108/7.18 million vaccinees) 1.6 (12 months)

0.6–4.0

Sturkenboom, 1999 (61)

USA

0.6 (2 months)

01–4.6

Weil, 1998 (62)

Confavreux et al., 2001 (54) Touze´ et al., 2000 (55) Sadovnick, 2000 (56)

0.4–4.8 0.3–3.0 0.4–2.1 0.7–1.7

Zipp, 1999 (57)

Verstraeten, 2001 (58) Coustans, 2000 (59) Fourrier, 2001 (60)

Abbreviations: MS, multiple sclerosis; OR, odds ratio.

calculation of relative risks. Of 643 patients with relapses of MS, 2.3% had been vaccinated during the preceding two-month risk period as compared with 2.8% to 4.0% who were vaccinated during one or more of the four control periods. The relative risk of relapse associated with exposure to any vaccination during the previous two months was 0.71 (95% CI, 0.40–1.26). There was no increase in the specific short-term risk of relapse associated with HB. Another recent study (53) also excluded a possible link between HB vaccine and MS. These authors conducted a nested case-control study in two large cohorts of nurses in the United States, those in the Nurses-Health Study (which has followed 121,700 women since 1976) and those in the Nurses-Health Study II (which has followed 116,671 women since 1989). For each woman with MS, five healthy women and one woman with breast cancer were selected as controls. The analyses included 192 women with MS and 645 matched controls. The multivariate relative risk of MS associated with exposure to the HB vaccine at any time before the onset of the disease was 0.9 (95% CI, 0.5–1.6). The relative risk associated with HB vaccination within two years before the onset of the disease was 0.7 (95% CI, 0.3–1.8). The results were similar in analyses restricted to women with MS that began after the introduction of the recombinant HB vaccine. These reassuring data are consistent with the fact that, since the integration of HB vaccine into national childhood immunization schedules in over 125 countries, it has been used in more than 500 million persons and has proved to be among the safest vaccines yet developed.

European multicenter study covering the period 1989 to 1994, the annual rate of increase in incidence was found to be 3.4%, with a particularly rapid rate of increase in children under four years (6.3%) (64). In this context, it is not surprising that the potential role of childhood vaccines as a triggering event for this disease has been questioned. This possibility has been evaluated in a few epidemiologic studies. A case-control study conducted in Sweden in the mid-1980s did not observe any significant effect of vaccination against tuberculosis, smallpox, tetanus, Pertussis, and rubella on diabetes (65). However, some authors (66) have hypothesized that the timing of vaccination may be of importance and that certain vaccines (e.g., Haemophilus influenzae type b, Hib), if given at two months of life or later might increase the risk of type 1 diabetes. This was not confirmed by a 10-year follow-up study of over 100,000 Finnish children involved in a clinical trial of Hib vaccine (Fig. 1). There was no increased risk

VACCINATION AND DIABETES Type 1 diabetes (formerly known as insulin-dependent diabetes mellitus, IDDM, or juvenile diabetes) results from autoimmune destruction of pancreatic b-cells in genetically susceptible individuals exposed to environmental risk factors. The incidence is particularly high in some geographic areas, for example, Finland and Sardinia, where it can reach 40 cases per 100,000. During the past decades, there was a regular increase of the incidence of type-1 diabetes in most countries of the world. In a recent

Figure 1 Cumulative incidence of type 1 diabetes per 100,000 person years in Finnish children aged 10 years or under. Comparison of children vaccinated first at the age of three months with children first vaccinated at the age of 24 months. Source: From Ref. 67.

Chapter 24: Vaccination and Autoimmunity

of diabetes when comparing children who had received four doses of vaccine at 3, 4, 6, and 14 to 18 months of age with children who received only one dose at 24 months of age (67). A recent study conducted in four large health maintenance organizations (HMOs) in the United States did not observe any association between receipt of routine childhood vaccines and the risk of type 1 diabetes. There was no influence of the timing of HB or Hib vaccination on the diabetes risk (68). Therefore, at this stage there are no serious indications of any significant influence of current childhood vaccines on the occurrence of type 1 diabetes.

NEW GENERATION VACCINES AND AUTOIMMUNITY: APPROACHES TOWARD EARLY RISK ASSESSMENT During the course of vaccine development, only a comprehensive and multidisciplinary strategy may help to reduce the theoretical risk that a new vaccine would induce autoimmune manifestations. First, one should question whether clinical manifestations of an autoimmune nature are known to be associated with the infectious disease that will be the target of the new vaccine. If such events have been reported, for example, for group A streptococcal diseases, attention should be given to avoid reproducing the natural disease pathogenic process. This may include the identification and the exclusion of naturally pathogenic epitopes. Second, potential molecular and immunological mimicry between vaccine antigens and host components should be extensively and critically analyzed through an intelligent combination of bioinformatics and immunological studies. One should keep in mind that, by itself, an identified mimicry is of little pathogenic significance. Information should be gathered on the relative ability of such epitopes to bind to human MHC molecules, to be processed by human antigen-presenting cells and to be recognized by autoreactive T cells. Molecular mimicry in itself is not sufficient to trigger autoimmune pathology and other factors intrinsic to infections such as tissue damage, and long-lasting inflammatory reaction might be required as well. For example, a recently developed Lyme disease vaccine was shown to contain an immunodominant epitope of the outer surface protein A of Borrelia burgdorferi (Osp A) displaying significant homology with human LFA-1, an adhesion molecule of the b2 integrin family. Although this raised concern about the safety of this vaccine, there was no evidence for an increased incidence of arthritis in individuals having received the Lyme vaccine (42). Third, indicative information can be obtained through the use of ad hoc experimental models of autoimmune diseases. Different vaccine formulations and adjuvants can be compared regarding their potential capacity to induce or enhance the expression of pathology in relevant models. For example, there are models of experimental allergic encephalitis, which are sensitive to the administration of IL-12 inducing microbial products and can help to compare the nonspecific effects of different adjuvants or vaccine formulations (32). Fourth, appropriate immunological investigations (e.g., autoimmune serology) may be systematically included in phase III-III clinical trials. On an ad hoc basis, clinical surveillance of potential autoimmune adverse effects may have to be included in the monitoring protocol. Such surveillance will have to be extended through the post-marketing stage if specific rare events have to be ruled out.

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CONCLUSION Isolated case reports and increased attention in the media to possible side effects of vaccines have dramatically modified the perception by the medical community and the public of the risk of autoimmunity elicited by vaccination, despite the lack of epidemiological support for such a concern. Although available data are reassuring, vigilance is still required as the risk of autoimmunity associated with some of the new generation vaccines might be increased as compared to current vaccines. A number of new adjuvants that are developed aim to induce strong Th1-type or Th17-type immune responses against viruses or other intracellular pathogens. Such effects may occasionally favor the expression of underlying autoimmune diseases or induce autoimmune responses in exceptional cases when the vaccine antigens do contain immunodominant epitopes that cross-react with self-antigens. Special attention should be given to adjuvants acting as strong inducers of IL-12 and IL-23 synthesis (30,31). Cancer vaccines based on dendritic cells pulsed with tumor antigens might also induce autoimmunity (69,70). There is an increasing interest in the combination of vaccines with agents targeting regulatory T cells or molecules involved in suppression of T-cell responses such as CTLA4 and PD1 (71,72). Clearly, this type of combined treatment will carry a significant risk of precipitating autoimmune pathology (73). Finally, it is of paramount importance to keep in mind that the mere occurrence of autoimmune markers (autoreactive antibodies or T cells) is a frequent phenomenon in a normal population and that pathological expression, that is, the development of an autoimmune disease, is by far much less frequent.

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37. den Haan JM, Kraal G, Bevan MJ. Cutting edge: lipopolysaccharide induces IL-10-producing regulatory CD4þ T cells that suppress the CD8þ T cell response. J Immunol 2007; 178:5429–5433. 38. WHO Global Advisory Committee for Vaccine Safety. Causality assessment of adverse events following immunization. Wkly Epidemiol Rec 2001; 76:85–89. 39. Schonberger LB, Bregman DJ, Sullivan-Bolyai JZ, et al. GuillainBarre syndrome following vaccination in the National Influenza Immunization Program, United States, 1976–1977. Am J Epidemiol 1979; 110:105–123. 40. Safranek TJ, Lawrence DN, Kurland LT, et al. Reassessment of the association between Guillain-Barre syndrome and receipt of swine influenza vaccine in 1976–1977: results of a two-state study. Expert Neurology Group. Am J Epidemiol 1991; 133:940–951. 41. Lasky T, Terracciano GJ, Magder L, et al. The Guillain-Barre syndrome and the 1992–1993 and 1993–1994 influenza vaccines. N Engl J Med 1998; 339:1797–1802. 42. Chen RT, Pless R, Destefano F. Epidemiology of autoimmune reactions induced by vaccination. J Autoimmun 2001; 16:309–318. 43. Miller E, Waight P, Farrington CP, et al. Idiopathic thrombocytopenic purpura and MMR vaccine. Arch Dis Child 2001; 84:227–229. 44. Vlacha V, Forman EN, Miron D, et al. Recurrent thrombocytopenic purpura after repeated measles-mumps-rubella vaccination. Pediatrics 1996; 97:738–739. 45. Oski FA, Naiman JL. Effect of live measles vaccine on the platelet count. N Engl J Med 1966; 275:352–356. 46. Jonville-Bera AP, Autret E, Galy-Eyraud C, et al. Thrombocytopenic purpura after measles, mumps and rubella vaccination: a retrospective survey by the French regional pharmacovigilance centres and pasteur-merieux serums et vaccins. Pediatr Infect Dis J 1996; 15:44–48. 47. Beeler J, Varricchio F, Wise R. Thrombocytopenia after immunization with measles vaccines: review of the vaccine adverse events reporting system (1990 to 1994). Pediatr Infect Dis J 1996; 15:88–90. 48. Nieminen U, Peltola H, Syrjala MT, et al. Acute thrombocytopenic purpura following measles, mumps and rubella vaccination. A report on 23 patients. Acta Paediatr 1993; 82:267–270. 49. Pool V, Chen R, Rhodes P. Indications for measles-mumps-rubella vaccination in a child with prior thrombocytopenia purpura. Pediatr Infect Dis J 1997; 16:423–424. 50. Gout O, The´odorou I, Liblau R, et al. Central nervous system demyelination after recombinant hepatitis B vaccination report of 25 cases. Neurology 1997; 48(Suppl.): A424 (abstract). 51. Gout O, Lyon-Caen O. Sclerotic plaques and vaccination against hepatitis B. Rev Neurol (Paris) 1998; 154:205–207. 52. Tourbah A, Gout O, Liblau R, et al. Encephalitis after hepatitis B vaccination: recurrent disseminated encephalitis or MS? Neurology 1999; 53:396–401. 53. Ascherio A, Zhang SM, Hernan MA, et al. Hepatitis B vaccination and the risk of multiple sclerosis. N Engl J Med 2001; 344:327–332. 54. Confavreux C, Suissa S, Saddier P, et al. Vaccinations and the risk of relapse in multiple sclerosis. Vaccines in Multiple Sclerosis Study Group. N Engl J Med 2001; 344:319–326. 55. Touze´ E, Gout O, Verdier-Taillefer MH, et al. The first episode of central nervous system demyelinization and hepatitis B virus vaccination. Rev Neurol (Paris) 2000; 156:242–246. 56. Sadovnik AD, Scheifele DW. School-based hepatitis B vaccination programme and adolescent multiple sclerosis. Lancet 2000; 355:549–550. 57. Zipp F, Weil JG, Einhaupl KM. No increase in demyelinating diseases after hepatitis B vaccination. Nature Medicine 1999; 5:964–965. 58. Verstraeten T, DeStefano F, Jackson L, et al. VSD Team. Risk of demyelinating disease after hepatitis B vaccination—West Coast, United States, 1995–1999. Paper presented at the 50th Annual Epidemic Intelligence Service Conference, 2001, Atlanta GA. 59. Coustans M, et al. Demyelinating disease and hepatitis B vaccination: Survey of 735 patients seen at MS clinic. Neurology 2000; 54 (Suppl. 3):A165–6, abstract.

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25 Adjuvants for the Future Richard T. Kenney Clinical R&D and Medical Affairs, Vaccines for Virus Diseases, North America GlaxoSmithKline Biologicals, Columbia, Maryland, U.S.A.

Alan S. Cross Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION The past decade has seen dramatic progress in our understanding of immune mechanisms and host defense. Along with the ability that vertebrates have to acquire immunity to pathogenic antigens by expanding specific populations of T and B cells and making cytokines and antibodies, scientists have discovered that like invertebrates, we have multiple innate pathways to activate more generic host responses through a whole new family of receptors. Over 80 years ago, Ramon demonstrated that it was possible to artificially increase antigen-specific levels of diphtheria or tetanus antitoxin by the addition of bread crumbs, agar, tapioca, starch oil, lecithin, or saponin to the vaccines (1). Since then, aluminum salts have been the dominant substance used and are still the only adjuvant currently used in licensed vaccines in the United States. The field has become much more sophisticated recently with the introduction of numerous new adjuvants and new concepts regarding the mechanisms of action. In this brief chapter, we review the modern adjuvants used in a variety of current and experimental human vaccines. After a more general discussion of adjuvants, including their definition, mechanisms of action, and safety, we will discuss recent clinical trials of investigational adjuvants. For additional study of this complex subject, including a historical perspective, the reader is referred to published reviews of vaccine adjuvants (see Refs. 2–4).

DEFINITIONS The term ‘‘adjuvant’’ (from the Latin adjuvare, meaning to help) was coined in 1926 by Ramon for a substance used in combination with a specific antigen that produces a stronger immune response than the antigen could if used alone (5). The enormous diversity of compounds, which increase specific immune responses to an antigen and thus function as vaccine adjuvants, makes any classification system somewhat arbitrary. Adjuvants can be loosely categorized in terms of their source or their physical nature as (i) mineral salts; (ii) mycobacterial, bacterial, and plant derivatives; (iii) surface-active agents and microparticles; (iv) polymers, cytokines, vitamins, and hormones; and (v) synthetic constructs. Those listed in Table 1 are examples of immunopotentiators used during the past 25 years. They are grouped according to origin rather than mechanism of action, because the latter are incompletely understood for

most adjuvants. All agents in Table 1 have immunomodulating capabilities and are reported to augment the immune response to specific antigens; nonspecific enhancers of the immune response that principally stimulate innate immunity are largely excluded. A comprehensive list of adjuvants, beyond the scope of this chapter, is available and updated by the NIAID (National Institutes of Health/National Institute of Allergy and Infectious Diseases, U.S.A.) (6). A ‘‘carrier’’ is an immunogenic protein to which a hapten or a weakly immunogenic antigen is bound (7). It may also be a living organism (or vector) bearing genes for expression of the foreign hapten or antigen on its surface. A naked DNA vaccine is a carrier in the sense that it entails injection into the host of a plasmid-based DNA vector that encodes the production of the protein antigen (8). Carriers increase the immune response by providing T cell help to the hapten or antigen. A ‘‘vehicle’’ provides the substrate for the adjuvant, the antigen, or the antigen-carrier complex. Unlike the carriers listed in Table 1, vehicles are not themselves immunogenic. Like carriers, most vehicles can enhance the immune response to antigens alone and are sometimes considered to be another class of adjuvants. Scientists have also investigated the result of combining adjuvants with different sources/mechanisms of action to increase their immunostimulatory effect. These ‘‘adjuvant formulations’’ can, in some cases, combine delivery improvement and immune modulation. Thus, in most cases, an adjuvant formulation is composed of two or more adjuvants with complementary immunomodulating effects, such as the adjuvant systems (ASs) being developed by GlaxoSmithKline (GSK). Many examples of such adjuvant formulations have been tested in humans.

MECHANISMS OF ACTION The effects of adjuvants can be strongly impacted by (i) the nature and dose of the immunogen; (ii) the nature and dose of the adjuvant(s) or carrier in the formulation; (iii) the stability of the formulation; (iv) the immunization schedule; (v) the route of administration; (vi) the species of animal; and (vii) the genetic and other biologic variations within species, including their immune status. The discovery of a class of receptors similar to Toll, an essential receptor for innate defense against fungal infection in Drosophila, changed the approach to understanding

Chapter 25: Adjuvants for the Future

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Table 1 Classes of Modern Vaccine Adjuvants, Carriers, and Vehicles Adjuvants Mineral salts Aluminum hydroxide (AlhydrogelTM; RehydragelTM), aluminum and calcium phosphate gel

Cytokines, vitamins, and hormones GM-CSF, IFN-g, IL-1, IL-2, IL-6, IL-7, IL-12, cytokinecontaining liposomes, vitamin A, D3 (calcitriol), E, human growth hormone, DHEA

Mycobacterial, bacterial, and plant derivatives

Surface-active agents and microparticles TM

Polymers

Complete Freund’s adjuvant (killed M. tuberculosis), DETOXTM (MPL plus cell wall skeleton of Mycobacterium phlei), BCG, muramyl dipeptides and tripeptides, dipalmitoyl phosphatidyl ethanolamine-MTP (MTP-PE), MPL, g-inulin/aluminum salts (algammulin), Klebsiella pneumoniae glycoprotein, Bordetella pertussis, Corynebacterium parvum, cholera toxin, E. coli LT, QS-21

Saponin (Stimulon QS-21, Quil-A), immune-stimulating complexes (ISCOMSTM), AvridineTM, nonionic block copolymers (CRL1005, pluronic L121), virosomes, DDA

Dextran, double-stranded polynucleotides (Poly rA:Poly rU), acetylated polymannose (Acemannan), sulfolipopolysaccharide, PMMA, acrylic acid-allyl sucrose (Carbopol), polyphosphazene (AdmumerTM), b-glucan (pleuran, algal glucan)

Synthetic constructs

Carriers

Vehicles

Imidazoquinolines (imiquimod, S-28463), glycolipid Bay R1005, stearyl tyrosine DTP-GDP (ImmTherTM) DTP-DPP (TheramideTM) Threonyl-MDP 7-allyl-8-oxoguanosine (Loxoribine) Multiantigen peptide (MAP) system Linear polymerization of haptenic peptides Peptide linkage to T cell or B cell epitopes

Bacterial toxoids (tetanus, diphtheria, Pseudomonas aeruginosa A exotoxin, pertussis), Meningococcal outer membrane proteins (proteosomes), Fatty acids, Ty virus like particles, Nucleic acid vaccines, living vectors (vaccinia virus, adenovirus, canarypox, poliovirus, BCG, attenuated Salmonella, Vibrio cholerae, and Shigella), protein cochleates

Mineral oil (IFA, MontanideTM; Specol) and vegetable oil (peanut, olive, sesame) emulsions, squalene and squalane emulsions (MF59, SAF, SPT), lipid containing vesicles (liposomes, DMPC, DMPG), sendai proteoliposomes, virosomes (IRIV), biodegradable polymer microspheres (lactide and glycolide polymers (PLGA, PGA, PLA), proteinoid microspheres (PODDSTM), polyphosphazene, protein cochleates, transgenic plants, chitosan polysaccharide

Abbreviations: BCG, bacillus Calmette-Gue´rin; PMMA, polymethyl methacrylate; DHEA, dehydroepiandrosterone; DDA, dimethyl dioctadecyl-ammonium bromide; MPL, monophosphoryl lipid A; LT, heat-labile toxin; GM-CSF, granulocyte-macrophage stimulating factor; IL, interleukin.

adjuvants over the past decade. Mammalian homologues or tolllike receptors (TLRs) have been discovered that provide a means to activate immune cells such as macrophages and dendritic cells with microbial ‘‘danger signals’’ (9). Bacterial lipopolysaccharide (LPS), which has long been known to be inflammatory if injected in very small quantities or if present as a trace contaminant in vaccines, was found to be a ligand for the TLR4 receptor (10). Since then, lipopeptides, flagellin, single- and doublestranded RNA, and CpG DNA found principally in bacteria and viruses have been linked to individual TLRs and provide a scientific basis for the development of adjuvants (11–13). However, many existing adjuvants may not function via the TLRs, and their mechanisms remain unknown (14). It is known that adjuvants can select for or modulate humoral or cell-mediated immunity, and they do this in several ways. First, antigen processing can be modified, leading to vaccines that can elicit both helper T cells and cytotoxic lymphocytes (CTLs) (reviewed in Ref. 15). Second, depending upon the adjuvant, the immune response can be stimulated in favor of type 1 or type 2 immune responses (16). For example, complete Freund’s adjuvant and the QS-21 adjuvant can elicit DTH and MHC class I CTL responses when mixed with protein antigens, peptides, or inactivated viruses (17). Many other adjuvants, such as aluminum salts (16) and nonionic block polymers (18) elicit principally antibody responses when combined with protein

antigens or inactivated organisms, perhaps by activating APCs by an IL-4-dependent mechanism (19). Third, adjuvants can augment the immune response by preferentially stimulating Th1 or Th2 CD4þ T-helper cells (20). The Th1 response is accompanied by secretion of interleukin-2 (IL-2), interferon-g (IFN-g), and TNF-a leading to a CMI response, including activation of macrophages and CTL and high levels of IgG2a antibodies in mice. The Th2 response is increased by secretion of IL-4, IL-5, IL-6, and IL-10, which provide better help for B cell responses, including those of IgG1, IgE, and IgA isotypes in mice. Aluminum salts and MF59 principally stimulate the Th2 response (21,22), while the Th1 response is stimulated by many adjuvants, such as muramyl dipeptide, monophosphoryl lipid A (MPL), and QS-21 (3). Vaccine adjuvants can modulate antibody avidity, specificity, quantity, isotype, and subclass against epitopes on complex immunogens (23–26). For example, only certain adjuvants, vehicles, and adjuvant formulations can induce the development of the protective IgG2a antibody isotype against Plasmodium yoelii (27), a mouse model of malaria. While an adjuvant’s effect on immunogenicity can be studied preclinically in animals, the models do not always anticipate their level of efficacy or safety in humans (28,29). However, decades of basic cellular research, preclinical experiments, and clinical safety and immunogenicity studies have led to a significant expansion in the understanding of the mode of

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action of many adjuvants. This is beginning to allow for rational design and combination of the optimal adjuvants for a particular antigen in a specific population, which can lead to safer and more effective vaccines.

SAFETY During the past 75 years, many adjuvant compounds have been studied, but most were never accepted for routine vaccination because of their immediate toxicity and fear of delayed side effects. The current attitude regarding the risks and benefits of vaccination favors safety over efficacy when a vaccine is given to a healthy population of children and adults (30). In high-risk groups, including patients with cancer and AIDS, and for therapeutic vaccines, an additional level of toxicity may be acceptable when the benefit of the vaccine is substantial. However, the absolute safety of any vaccine cannot be guaranteed, so the risks must be minimized. Undesirable reactions can be grouped as either local or systemic. The most frequent local adverse effects of vaccination are tenderness and swelling, with the most severe ones involving the formation of painful induration and nodules at the inoculum site. The mechanisms for such severe local reactions include formation of inflammatory immune complexes at the inoculation site by combination of the adjuvanted vaccine with preexisting antibodies resulting in an arthus-type reaction. In some cases, poor biodegradability of the adjuvanted vaccine may result in prolonged persistence in the tissues and reactive granuloma formation. Such local reactions are of concern for depot-type adjuvants and living vectors such as BCG. Those adverse effects are rare events with today’s vaccines. Clinical studies typically reveal this type of problem before a vaccine is licensed and development is halted. Severe local reactions in humans followed subcutaneous injections of incomplete Freund’s adjuvant (IFA), a mineral oil emulsion, using early formulations made with a mannide monooleate stabilizer that contained free fatty acid impurities. However, these lesions did not occur with IFA injected intramuscularly, and that contained the stabilizer without impurities (reviewed in Refs. 28 and 31). IFA has been administered to more than a million people worldwide (31–34). Despite the apparent long-term safety of this adjuvant (35), the risk/benefit ratio is felt to be too high for commercial use. To date, vaccine adjuvants have caused few severe acute systemic adverse effects. More theoretical risks include the induction of autoimmunity or cancer. Fortunately, in 10-, 18-, and 35-year follow-up studies, the incidence of cancer, autoimmune and collagen disorders in 18,000 persons who received the IFA adjuvanted influenza vaccine in the early 1950s was not different from that in persons given aqueous vaccines (32,35– 37). Autoimmunity can be triggered by an infection through either specific or nonspecific mechanisms, although this has been associated with vaccination only in rare circumstances, such as when a form of Guillain-Barre´ syndrome was linked to the 1976 to 1977 vaccination campaign against swine influenza (38). Extensive epidemiological studies have failed to show an association of autoimmune disease with vaccination in nearly all instances (39). Studies in animals can provide signals that would lead to further study. Anterior chamber uveitis has been reported with MDP and several MDP analogues in rabbits (40) and monkeys (41), and has been systematically sought in at least one adjuvant vaccine study involving 110 volunteers, but was not detected (42). Adjuvant-associated arthritis (43,44) has not been reported in humans, even after long-term follow-up

(33,35,45). Anaphylactic reactions, angioedema, urticaria, and vasculitis have been described following the administration of the majority of vaccines, although severe events are rare (29). Finally, a syndrome known as macrophagic myofasciitis (MMF), characterized by diffuse arthromyalgias and fatigue in connection with muscle infiltration by macrophages and lymphocytes, has been described in France (46), although a causal association with vaccination has not been established.

REGULATORY ISSUES In concert with the progress of the International Conference on Harmonisation (ICH) of technical requirements for registration of pharmaceuticals for human use, worldwide regulatory guidance on the development and testing of vaccines has expanded significantly in the past few years. Documents covering nearly every aspect of drug and biologic development are being created and revised in an effort to enhance and standardize the quality, safety, and efficacy of pharmaceutical products (http://www.ich.org, http://www.fda.gov/cber/guidelines. htm, and http://www.emea.eu.int). There is little advice directed specifically at the development of adjuvants in the United States, apart from their use in combination vaccines (47). The European Medicines Agency (EMEA) recently published a guidance that emphasizes a number of additional points: quality of manufacture of the adjuvant alone and in combination with vaccine antigens, nonclinical proof of concept and toxicity studies, and clinical development that assesses the adjuvant effect and dose required (48). It is important to note that as a rule, adjuvants are not licensed on their own. Since each combination of one or more antigens with an adjuvant has its own unique safety and efficacy profile, they are licensed and regulated as individual vaccine products in combination.

ADJUVANTS USED IN LICENSED VACCINES FOR HUMANS Several adjuvants are licensed with their vaccines for human use in various parts of the world, including aluminum compounds, MF-59, virosomes, exotoxins, and AS04.

Aluminum Compounds Aluminum salts, particularly aluminum hydroxide or phosphate, have been used for over 80 years (49) and have become the most widely used adjuvants in human vaccines. Vaccine antigens can be adsorbed to the amorphous crystalline gel by electrostatic interactions between proteins and the positively charged aluminum hydroxide. Alternatively, negatively charged aluminum phosphate gels can bind proteins through a ‘‘ligand exchange’’ between hydroxyl and phosphate groups (21). Calcium phosphate has also been used to adsorb DPT, inactivated polio vaccines, and allergens (50). The following licensed, parenterally administered human vaccines are combined with aluminum: diphtheria, pertussis, and tetanus alone or in various combinations with Haemophilus influenzae type b (Hib), inactivated polio, hepatitis B, hepatitis A, a rabies vaccine, an anthrax vaccine, and GardasilTM a human papillomavirus vaccine recently licensed by Merck. The major advantage of using aluminum adjuvants is their safety record after billions of doses, and the development of earlier, higher, and longer-lasting antibody after primary immunization compared to primary immunization with soluble vaccines, particularly of soluble toxoids, although the

Chapter 25: Adjuvants for the Future

aluminum-adsorbed vaccines do not show any advantage over soluble preparations for booster responses (51). While aluminum adjuvants can stimulate Th2 type responses in mice and the production of cytokines such as IL-4 and IL-5, as well as B cell production of IgG1 and IgE, they fail to stimulate Th1 responses such as IFN-g production and B cell IgG2a secretion. The mechanism of adjuvanticity is still a subject of debate and includes formation of a depot at the injection site allowing slow release of antigen, stimulation of immunoreactive cells via activation of complement, activation of macrophages, and efficient uptake of aluminum-adsorbed antigen particles by antigenpresenting cells because of their particulate nature and optimum particle size (<10 mg) (16,51). The limitations of aluminum adjuvants include (i) the potential for induction of occasional painful nodules or swelling and erythema at the inoculation site, and the induction of antigen-specific IgE antibody that correlates with such local reactions (51,52), although the incidence of systemic immediate hypersensitivity is probably less than one in a million (53). (ii) Aluminum has been detected at the site of subcutaneous injections for up to one year in animals (51), so it is not readily ‘‘biodegradable.’’ In addition, the aluminum compounds have several immunological drawbacks including (iii) their inability to enhance humoral immunity against certain vaccines in humans such as typhoid (54), influenza hemagglutinin antigen (55), and Hib capsular polysaccharide-tetanus toxoid conjugate (56), and (iv) their near total inability to elicit cell-mediated immune responses, particularly cytotoxic T-cell responses to intracellular organisms (16). Finally, (v) careful formulation of aluminum adjuvant preparations is required for reproducibility, they cannot be sterilized by filtration, and they cannot be frozen or readily lyophilized (51).

Microfluidized Oil/Water Emulsion (MF59) A series of squalene emulsions were prepared using a microfluidizer to generate small particle (200–300 nm), oil-in-water (O/W) emulsions that had low-viscosity and were biodegradable (57). The most stable emulsion, termed MF59, consists of 4.3% (vol/vol) squalene and 0.5% (vol/vol) each of the surfactants Tween 80 (polyoxyethylene sorbitan monooleate) and Span 85 (sorbitan trioleate). Overall, MF59 generates antibody titers consistently higher than those obtained with aluminum hydroxide, equal to or higher than IFA, and equal to or lower than CFA, although it does not stimulate antibody responses against squalene (58). Results of timed injection studies suggest that MF59 microdroplets activate the immune system in the absence of antigen. It is postulated that macrophage uptake of the emulsion droplets results in cytokine production, which leads to an enhanced immune response in the presence of the antigen (57). MF59 has been tested in a variety of animal species, showing a good safety profile and a significant increase of the immune response to several subunit antigens including CMV, HSV, HIV, HCV, HBV, and influenza antigens. Novartis Vaccines (formerly Chiron Biocine, Siena, Italy) registered an influenza vaccine adjuvanted with MF59 as FLUADTM in much of Europe, which has been given to more than a million people (59). The MF59 formulation has also been tested in combination with pandemic influenza antigens, recombinant HSV glycoproteins, hepatitis B virus PreS2/S antigens, and HIV envelope proteins with various degrees of success (22). Study populations have included healthy adults (HSV, HBV, HIV, influenza) (60), elderly populations (influenza) (61), and

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infants and children (HIV) (57). Overall, MF59 has had acceptable reactogenicity profiles, although in the NIH comparison trial of multiadjuvanted HIV gp 120 vaccine described above, MF59 þ MTP-PE (in addition to SAF þ threonyl-MDP) induced significantly more moderate to severe local reactions than did other adjuvants (62).

Virosomes Immunopotentiating reconstituted influenza virosomes (IRIV) are 150 nm unilamellar vesicular proteoliposomes composed of influenza H1N1 surface glycoproteins intercalated in a mixture of natural and synthetic phospholipids (63). The influenza HA antigen binds to sialic acid on the surface of antigen-presenting cells that take up the particles by receptor-mediated endocytosis and, subsequently, by pH induced membrane fusion with the phagolysosomal membrane. IRIV can act as antigen carriers to deliver many types of antigens bound or conjugated to the surface or internalized. Given the unique properties of the system, after proteolytic degradation, the antigenic peptides can become complexed with both MHC class I and class II molecules to be expressed on the surface of the APC. The initial application of this system was with a virosomal hepatitis A vaccine. Berna Biologics, Ltd. (now owned by Crucell, Leiden, The Netherlands) registered EpaxalTM in several European, Asian, and South American countries after clinical testing, which showed an acceptable immune response and a significant reduction in local reactions compared to the conventional alumadsorbed vaccine (64,65). A second example, Inflexal VTM (Solvay Pharmaceuticals, Brussels, Belgium), is a trivalent influenza vaccine that is made by mixing three monovalent virosomes, each one containing the seasonal HA and NA glycoproteins recommended annually by WHO (63). This technology was licensed by Solvay, and their virosomal influenza vaccine has been marketed as InvivacTM since 2004, showing similar immunogenicity to FLUAD and decreased reactogenicity (66). The virosome system is being further developed for use with a DPT vaccine, as well as other antigens.

Exotoxins The bacterial ADP-ribosylating exotoxins (bAREs) represent a potent group of proteins that have been studied as enteric, nasal, and topical adjuvants for decades, and this category includes both licensed (albeit since withdrawn) and experimental vaccines. The only licensed vaccine that included a bARE as adjuvant was the intranasal virosome-based influenza vaccine that included a low dose of the E. coli heat-labile toxin (LT) for mucosal immunization (61,67). In pre-licensure trials, the vaccine was well-tolerated and elicited secretory IgA mucosal responses to influenza hemagglutinin, as well as serum antibody responses (68,69). However, post-licensure surveillance indicated that the vaccine was associated with an increased occurrence of Bell’s palsy, and it was concluded that the intranasal administration of wild-type LT was likely to be an important contributing factor (70,71). Interestingly, extensive preclinical toxicology studies did not predict such adverse reactions (72).

AS04 GSK Biologicals has been developing novel AS for more than a decade. These are unique combinations of different compounds with immunomodulating abilities that can tailor an

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immune response to a specific disease and target population. One of these, AS04, is a combination of aluminum salt and 3-O-desacyl-40 -MPL, a purified, detoxified derivative of bacterial lipopolysaccharide. MPL acts through binding to TLR4, a toll-like receptor found on macrophages and dendritic cells, to enhance the secretion of inflammatory cytokines and chemokines. AS04 was combined with hepatitis B antigen to create FENDrixTM, which is approved in by the EMEA for the prevention of hepatitis B in high-risk pre-hemodialysis and hemodialysis patients over 15 years. Clinical trials demonstrated that the hepatitis B/AS04 vaccine resulted in higher antibody levels, enhanced cell-mediated immunity and increased rates of seroprotection compared to a classical hepatitis B vaccine adjuvanted with aluminum salt alone (73). In addition, a cervical cancer vaccine called CervarixTM, which is formulated with human papillomavirus antigens as virus-like particles (VLPs) of type 16 and 18 and adjuvanted with AS04, was recently approved in Australia and has been submitted for licensure in several other areas of the world (74–76).

EXPERIMENTAL ADJUVANTS IN HUMANS

Table 2 Selected Clinical Trials of Experimental Adjuvanted Vaccines Against Infectious Diseases in the Past Decade Vaccine Viral CMV HCV Hepatitis B

Herpes simplex gp’s HIV-1 gp120/gp160 HIV NefTat/gp120 HIV gp120W61D HIV p17/p24 HIV-1 inact. HIV TAB9 HPV

The number of commercially feasible adjuvants tested in animals and humans (Table 1) is too large to review in this short chapter. Instead, a smaller number of modern adjuvants or adjuvant formulations used to enhance a variety of experimental vaccines in humans (Table 2) will be considered. The development of experimental adjuvants has been driven principally by the failure of aluminum compounds to (i) enhance many vaccines in man (31), (ii) enhance subunit vaccine antigens in animals (28,144,145), and (iii) to stimulate cytotoxic T-cell responses (146). In many instances, several adjuvants have been combined in one adjuvant formulation, hoping to obtain a synergistic or additive effect.

Influenza

Emulsion-Based Formulations

S. pneumoniae Tuberculosis

Two basic concepts have emerged in the manufacture of aqueous and oil combinations that describe the dispersion of one liquid as particles within a second liquid that is continuous (147). Surfactants, which are compounds that contain both polar and nonpolar groups, are added to stabilize the emulsions. Their hydrophilic/lipophilic balance determines the state of the emulsion that forms. Water-in-oil (W/O) emulsions that Freund used were initially very unstable and viscous and caused strong local reactions, yet they are very efficient at inducing an immune response to weak antigens. Newer oils and surfactants are now used, which allow the development of stable fluid emulsions that are safer (147). Mineral oils in W/O emulsions, such as IFA, stay at the injection site, and are slowly eliminated by macrophages or metabolized to fatty acids, triglycerides, phospholipids, or sterols (148). Protein antigens are released very slowly from this matrix. Proprietary, highly refined emulsifiers from the mannide monooleate family in a natural metabolizable oil solution were developed by SEPPIC (Paris, France), named MontanideTM ISA 51 and ISA 720. Both Montanide adjuvants induce a strong immune response, but severe local reactions may limit their use. O/W preparations containing small particles of oil dispersed in an aqueous continuous phase are more easily cleared from the injection site. The droplets with antigen can be endocytosed by APCs or readily pass from the injection site

Influenza, pandemic

Bacterial Anthrax rPA Clostridium difficile Diphtheria toxin ETEC Borrelia OspA

Parasitic Leishmaniasis

Malaria CS

Malaria blood stage

Vehicle, adjuvant, or formulation IL-12 (77) Poly-L-arginine (78) AS04 (79–82) RC-529 þ Al salts (83) DNA vaccination (84) Immunostimulatory oligos (85–87) MF59 (88) Flt3 ligand (89) MF59 (90) AS04 (91, 92) Al salts (93) MF59  Al salts (94) AS02 (95) AS02 (96) Ty-VLP (97) IFA (98) Montanide ISA 720 (99, 100) AS04 (75, 76) Al salts (101, 102) ISCOMS (103) LT (104) Virosomes or MF59 (61, 66, 105) Proteosomes (106, 107) Immunostimulatory oligos (108) MF59 (109, 110) Al salts (111–114) O/W emulsion-based AS (115) Al salts (116) Al salts (117) Chitosan (118) LT (119, 120) Al salts (121) rBCG (122) MPL þ Al salts (123) AS02 (124) BCG bacteria (125, 126) Alum/Inactivated L. major þ BCG (127) rAg þ GM-CSF (128) QS-21 þ Al salts (129) AS02 (119–137) Montanide ISA 720 (138–142) AS02 (131, 143)

Abbreviations: IL, Interleukin; ISCOMS, immunostimulatory complexes; LT, labile toxin; Al salts, aluminum salts; ETEC, enterotoxigenic Escherichia coli; rBCG, recombinant bacille Calmette-Gue´rin; MPL, monophosphoryl lipid A; BCG, bacille Calmette-Gue´rin; GM-CSF, granulocyte-macrophage stimulating factor.

to lymphatics (149). A major part of the effort to develop immunostimulators as vaccine adjuvants has been devoted to the characterization of mycobacterial cell wall components and their analogues as additions to these preparations (31,150,151). The most studied component of the cell wall has been the muramyl dipeptide, N-acetylmuramyl-L-alanyl-Disoglutamine (MDP). Three promising derivatives of MDP were developed because of its residual toxicity and pyrogenicity; they include a butyl-ether derivative (Murabutide TM ) (152,153), threonyl-MDP(57,154), and muramyl tripeptide

Chapter 25: Adjuvants for the Future

dipalmitoyl phosphatidylethanolamine (MTP-PE) (57,145). Because MDP in water provides only a modest adjuvant effect in mice (155,156) and humans (152), threonyl-MDP and MTPPE have been administered in oil emulsion vehicles in attempts to improve potency. The Syntex Adjuvant Formulation (SAF) preparation (Syntex Research, acquired by Roche in 1995) is an O/W emulsion vehicle. The vehicle contains 5% squalane, 2.5% PluronicTM L121, and 0.2% polysorbate 80 (TweenTM 80) in phosphate-buffered saline, pH 7.4 (149,157). Squalane, used in several modern adjuvant emulsions, is metabolizable oil used in many over-the-counter drugs and cosmetics. Pluronic 121 is a nonionic block copolymer discussed below. SAF elicits both cell-mediated (lymphocyte blastogenic) and humoral responses, but it is highly reactogenic so it is no longer studied as a vaccine adjuvant. GSK Biologicals is also developing a proprietary O/W emulsion-based AS for use in a prepandemic H5N1 influenza vaccine to provide heterologous immunity (115). Finally, MF-59 has been studied by Chiron in various experimental vaccines for HIV, herpes simplex, and HPV (57,90,94,158).

Monophosphoryl Lipid A The adjuvant effect of LPS was described in 1956 (159). Most of the adjuvanticity and toxicity of LPS are associated with the lipid A region of the molecule (160). The LPS of Salmonella minnesota R595 has been detoxified without destroying its adjuvant activity by exposing the LPS to mild hydrolytic treatment (161). The resultant monophosphoryl derivative of lipid A, called MPL, is a highly adaptable molecule that can be used effectively in many adjuvant formulations (162). The immunopotentiating nature of MPL may be associated with its capacity to induce cytokines such as IL-12 (163), IFN-g, IL-1, and IL-2 in mouse and human macrophages (164–166). MPL promotes antigen-specific DTH and a predominant murine IgG2a immunoglobulin response characteristic of TH1 help (167). Numerous animal and human studies testify to the utility of MPL as an adjuvant, used alone or combined effectively with other adjuvants and vehicles for capsular polysaccharide, protein, and peptide antigens (74,123,162). In the past decade, many clinical studies have utilized MPL or DETOXTM (MPL plus cell wall skeleton of Mycobacterium phlei in a squalane-inwater emulsion vehicle) as vaccine adjuvants in volunteers (75,76,79–82,95,119,123,130,131,143,168–170) (Table 2). More recently, synthetic lipid A mimetics (aminoalkyl glucosaminide 4-phosphates) that share most of the properties of MPL, have been developed by Corixa, and are now being developed by GSK (83,171,172). Several AS under development by GSK Biologicals contain MPL combined with O/W emulsions. AS02 (formerly known as SBAS2) is a proprietary O/W emulsion containing MPL and QS-21 that causes strong antibody responses as well as Th1 and CTL cellular responses. Phase 1/2 studies have been conducted in hepatitis (82), HIV (95), and with multiple HIV vaccine formulations (96). AS02 has been broadly studied in malaria, most recently with RTS,S, a circumsporozoite (CS) subunit antigen fused to the hepatitis S antigen (119,130,132–134), or with FMP1, a 42-kDa fragment of the merozoite surface protein-1 (131,143). RTS,S with AS02 demonstrated efficacy in Phase 2b field trials in The Gambia (168) and Mozambique (135–137). AS04 (described above) is comprised of aluminum salts and MPL for use in licensed vaccines. This AS has also been studied in HSV (173).

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Exotoxins Recombinant LT, which is one of the most potent mucosal adjuvants (174), has been shown to be safe and immunogenic by transcutaneous immunization (TCI) in humans (175). Antigens can be formulated with LT and delivered using a topical patch for delivery to the dense population of dendritic cells that are resident in the epidermis. LT-specific IgG and IgA antibodies were present in both stool and urine, implying the induction of a strong mucosal immune response. The potent activation of epidermal Langerhans cells allows LT to adjuvant the response to a coadministered enterotoxigenic Escherichia coli (ETEC) antigen as well (176). Serological and antibody-secreting cell (ASC) responses to the LT and the E. coli surface antigen CS6 were comparable to those seen following a protective oral challenge, suggesting that TCI can potentially elicit effective immunity similar to natural infection with ETEC, although local rashes may limit its broad applicability (176). LT is being studied as an immunostimulant to be used with TCI in conjunction with an injected influenza vaccine (104,177). Other groups are using detoxified mutants of LT to explore the potential for oral or intranasal vaccination (178–180), however, there is at least a theoretical concern that the LT could traffic to the brain and could cause inflammation there (72).

Saponins Saponins are triterpene glycosides that can be isolated from the bark of the Quillaja saponaria Molina tree, a species native to South America (181). A partially purified saponin, Quil A, has been used widely as an adjuvant in veterinary vaccines (182). Quil A is a heterogeneous mixture of glycosides. Analysis by high performance liquid chromatography (HPLC) reveals at least 24 peaks that vary in their adjuvanticity and toxicity in mice (183). Quil A has also been tested extensively as part of immune-stimulating complexes known as ISCOMsTM, which are colloidal cage-like 40 nm particles consisting of antigen, cholesterol, phospholipids, and Quil A (184). Despite their potent adjuvanticity, ISCOM vaccines have only recently been administered to humans because of the local and systemic toxicity of Quil A in mice (184,185). An influenza-ISCOM vaccine for humans containing a less toxic saponin fraction is under development, which shows a strong cellular immune response (103,186). QS-21 (StimulonTM) is one of at least 24 structurally distinct triterpene glycosides isolated from Quil A, and is being developed by the Antigenics, Inc. (Framingham, Massachusetts, U.S. ). It demonstrated the proper balance of low mouse toxicity and maximum adjuvanticity, and it eliminated the problem of lot-to-lot variation characteristic of Quil A (183). QS-21 is novel in that it can improve the immunogenicity of protein and polysaccharide antigens (187) in a variety of small animals, dogs, or primates. It also uniquely stimulates both humoral and cell mediated immunity, including potent class I-restricted cytotoxic T lymphocyte responses to subunit antigens (188). The addition of QS-21 to malarial peptide vaccines promoted CD4 and CD8 T cell responses (189). As noted above QS-21 is synergistic with MPL in stimulating the response to several vaccines in AS02.

Nonionic Block Copolymers The copolymer adjuvants are simple linear chains or blocks of polymers of hydrophobic polyoxypropylene, flanked by two

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chains of hydrophilic polyoxyethylene. A large number of copolymer adjuvants have been synthesized by varying the constituent chains (190). Nonionic block copolymers are currently used commercially in over-the-counter products, including shampoos, mouthwashes, and cosmetics. Copolymers are adhesive molecules that bind antigens to hydrophobic surfaces, such as oil drops or cells (191). Evidence suggests that proteins bound to copolymer are held firmly in a condensed fashion, and retain much of their native B-cell epitope confirmation when presented to macrophages and dendritic cells for immune processing (191). The activation of complement by contact with the copolymer surface augments the adjuvant effect. Several preparations of block polymers developed by Vaxcel, Inc. (Norcross, Georgia, U.S.) are awaiting clinical trial (190,192).

Cytokines The use of cytokines as vaccine adjuvants has been encouraged due to a better understanding of cytokine mechanisms and the commercial availability of recombinant interferon-g (IFN-g) and granulocyte-macrophage stimulating factor (GM-CSF). Many cytokines (e.g., IL-3, IL-6, IL-11, GM-CSF) are capable of enhancing various immune responses when administered repeatedly. But the cytokines with the greatest potential are those administered in a single dose at or near the time of antigen injection; cytokines administered in this practical way include IFN-a, IFN-g, IL-1, IL-2, IL-12, and GM-CSF. A cytokine can enhance, inhibit, or have no effect, depending on the dose, timing, and animal species, and which of these effects predominates is not always predictable (193,194). The adjuvant effects of these cytokines in animals or humans have been reviewed in detail (195–197), although trial results to date have failed to document a strong adjuvant effect for cytokines in humans (198). The cytokine most intensively studied for its adjuvant activity is GM-CSF. It has been used with hepatitis B vaccine in patients with chronic renal failure (199,200) as well as in patients with HIV infection (201). In each of these studies, it enhanced the vaccine response, but was usually administered 24 hours before the vaccine. In contrast, when given concurrently with hepatitis A, influenza, and tetanus-diphtheria toxoid vaccines, a lower response was observed (202), which emphasizes that the effect of the timing of GM-CSF administration requires further study. Nevertheless, two metaanalyses concluded that the use of GM-CSF with hepatitis B vaccines both accelerated and increased the response rate (203,204). GM-CSF has also been used with a leishmania vaccine (128). Another cytokine, flt3 ligand, enhances dendritic cell numbers and function, but this effect was not translated into an improved antibody response (89,205). The use of rIL-12 with an experimental vaccine for CMV improved both humoral and cellular immune responses in human subjects (77).

Immunostimulatory Oligonucleotides: CpGs Just as bacterial DNA can activate immune cells, synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides in particular base contexts (CpG motifs) stimulate the innate immune system to induce protection in mice and primates (206,207). Either alone or in combination with a vaccine, they can activate human B cells, DC, and NK cells (208) and trigger an immune cascade that includes the production of cytokines, chemokines, and IgM to protect against infection. CpG ODN are extremely efficient inducers of Th1 immunity and CTL, and can allow a 10- to 100-fold

reduction in the dose of antigen, presumably because of the increased efficiency of antigen presentation by DC (209). The administration of CpG ODN is currently being tested as a stand-alone treatment for cancer and asthma, and as a vaccine adjuvant (210–213). Acting through TLR9 receptors present on B cells and plasmacytoid dendritic cells, CpG has been shown in many studies to both accelerate and enhance the response to hepatitis B vaccines in human subjects (214,215) with protective and sustained levels of anti-hepatitis B surface antigen achieved with fewer doses of vaccine. The adjuvant improved the anti-HBs antibody levels in HIV patients (85), and enhanced the affinity of anti-HBs antibodies independently of the titers achieved (86). Immunostimulatory sequences have been used with hepatitis B vaccine proteins (87,214,216). While the addition of CpG ODN to hepatitis B vaccines has improved the immune response to this vaccine, no improvement was observed when added to an influenza vaccine (108). However, CpGs did enhance the response to that same vaccine when given at 1/10, the standard dose.

SUMMARY AND CONCLUSION Every adjuvant has a complex and often multifactorial immunological mechanism, usually poorly understood in vivo, although the discovery of the TLRs and mechanisms of innate immunity will help guide adjuvant development over the next decade. Adjuvant safety, including the real and theoretical risks of administering vaccine adjuvants to humans, is a critical component that can enhance or retard adjuvant development. In addition to the problem of safety, several other issues impede the orderly development of adjuvanted vaccines. These include inconsistent immunopotentiation by candidate adjuvants, marked variation in response to the same adjuvant by different animal models, and the inability to consistently predict protective efficacy by immunoassays. However, decades of basic cellular research, preclinical experiments, and clinical safety and immunogenicity studies have led to a significant expansion in understanding the role of adjuvants in recent years. Hopefully, this will open new doors in vaccine research. The most studied experimental adjuvants in man include aluminum compounds, oil-based emulsions with or without muramyl dipeptide, monophosphoryl (detoxified) lipid A, MPL, the triterpene glycoside QS-21, nonionic block copolymers, several cytokines, especially GM-CSF, and CpG ODNs. In preclinical studies of adjuvants and vaccines, depending on the antigen used, the same adjuvant can enhance, inhibit, or have no effect at all. The more important determinants of immunogenicity include the nature and dose of the immunogen, the schedule and route of administration, the population being immunized, the stability of the adjuvant formulation, and the choice of adjuvants used alone or in combination. The increasing understanding of these determinants is fundamental to the further development of new vaccines. This is beginning to allow for rational design and combination of the optimal adjuvants for a particular antigen in a specific population, which has the potential to lead to safer and more effective vaccines. In addition to immunologic enhancement without toxicity and successful protection against challenge, choice of adjuvant for a clinical trial may depend on cost and commercial availability. Rational development of classical and novel adjuvants will continue to be one of the most important challenges for the vaccinologist to be able to address persistent unmet medical needs.

Chapter 25: Adjuvants for the Future

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26 TLR9 Agonists for Immune Enhancement of Vaccines Heather L. Davis Pfizer Vaccines Research Unit, Ottawa, Ontario, Canada

INTRODUCTION This review of the use of CpG oligodeoxynucleotide (ODN) agonists to toll-like receptor (TLR)9 as vaccine adjuvants briefly covers immune effects and mechanisms of CpG ODN and then provides more details as to how they can be used to enhance prophylactic and therapeutic vaccines against human or veterinary infectious diseases, cancer, and allergies. Induction of adaptive immunity relies on simultaneous presentation of antigen to B and T cells, as well as activation of cells of the innate immune system including dendritic cells (DC), macrophages, and monocytes. Both B and T cells have highly specific receptors that recognize antigenic epitopes, and this in turn results in the development of antigen-specific antibodies and cytotoxic T-cell responses, respectively. In contrast, cells of the innate immune system lack highly specific antigen receptors but instead rely on a set of ‘‘pattern recognition receptors’’ (PRR), which have a general ability to detect ‘‘pathogen-associated molecular patterns’’ (PAMP) found in pathogens but not in self-tissues. Many of the PRR are found in the family of toll-like receptors (TLR), of which at least 10 types have been identified in humans. The immune system appears to use the presence of PAMP as a ‘‘danger signal’’ that indicates the presence of infection and activates appropriate defense pathways (1–3). Some TLR are located on the surface of immune cells and detect PAMP that would be present in the extracellular space. These include TLR2 and TLR6 that detect proteoglycans/peptidoglycans and bacterial lipopeptide (TLR2 only), TLR4 that detects lipopolysaccharide of gram-negative bacteria, and TLR5 that detects flagellin. Another group of TLR is located in the endosomal compartment of immune cells that detect nucleic acid–based PAMP that would be preferentially seen in the intracellular space. These include TLR3 that detects viral dsRNA, TLR7 and TLR8 that detect viral ss-RNA, and TLR9 that detects ‘‘CpG motifs’’ of bacterial and viral ssDNA (4). In some cases, the nucleic acid–binding TLR can also be activated by small-molecule mimics of their natural ligands, as is the case for imidazoquinolines that activate TLR7 and TLR8 (5,6); however, to date, there have been no reports of identification of small molecules that activate TLR9.

TOLL-LIKE RECEPTORS AND RECOGNITION OF CpG DNA BY TLR9 Recently there has been broad interest in testing and developing such danger signal ligands of PRR for immune stimulation, including use as adjuvants with vaccines to enhance antigenspecific responses. With respect to TLR9, synthetic ODN con-

taining CpG motifs (CpG ODN) are being developed as immune therapy drugs and vaccine adjuvants. There is an extensive literature regarding the molecular pattern in viral and bacterial DNA that activates TLR9, and the downstream signaling pathways. Since this chapter reviews the use of CpG ODN as vaccine adjuvants, only a brief outline on these aspects will be provided and more information can be obtained from other recent reviews (4). TLR9 is activated by CpG motifs that are unmethylated CpG dinucleotides within the context of certain flanking bases. These motifs are recognized as foreign, since mammalian DNA has suppression of CpG dinucleotides and the cytosine is usually methylated, which renders them nonimmune stimulatory. In humans, only B cells and plasmacytoid dendritic cells (pDC) express TLR9. Activation of other cell types results through indirect means, largely cytokine mediated. In mice the distribution of TLR9 is broader, including monocytes and myeloid-derived dendritic cells (mDC). CpG ODN enters immune cells after binding to cell surface DNA-binding proteins (non-sequence specific) and ends up within the endosomal compartment where it activates TLR9 (sequence dependent). There is some degree of species specificity with respect to optimal flanking sequences, with GACGTT being optimal in mice and GTCGTT being optimal in humans but also working in most species (4). Several different classes of CpG ODN have been described that differ largely in ability to form higher-ordered structures. These give different stimulatory profiles on human immune cells in vitro, but it is not clear whether these differences translate in vivo and in particular when used as vaccine adjuvants. Other factors that largely affect potency are the number and spacing of CpG motifs within the ODN and backbone modifications (4). Almost all vaccine data, heretofore, have been obtained with simple monomeric B-class CpG ODN.

NONCLINICAL STUDIES: GENERAL UTILITY OF CpG ADJUVANTS Many of the identified direct and indirect effects of CpG ODN on immune cells could contribute to its efficacy as a vaccine adjuvant. Humoral responses are augmented because of CpG activation of B cells to secrete immunoglobulin and cytokines, aided by cross talk between the B-cell receptor and CpG signaling pathways. CpG also induces increased costimulatory molecule expression on B cells and other antigen presenting cells (APC). Furthermore, CpG inhibits B-cell apoptosis, contributing to a more sustained immune response (7,8). The most unique feature of CpG as an adjuvant is its outstanding ability

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to induce Th1-dominated immune responses, similar to the ‘‘gold standard,’’ complete Freund’s adjuvant, as measured by its ability to drive the differentiation of cytotoxic T lymphocytes (CTL) and IFN-g secreting T cells. With purified protein antigens, unlike whole attenuated pathogens, the generation of CD8þ CTL is strongly CD4 dependent. An adjuvant such as CpG ODN that allows APC to prime CD8þ T-cell responses in the absence of T-cell help is very attractive for the new generation of therapeutic vaccines that depend on T-cell responses for efficacy. CpG have been shown to work virtually with any type of antigen, including recombinant proteins, polypeptides, and peptides, as well as virus-like particles, whole-killed pathogens, and live attenuated viruses. One exceptional situation is with polysaccharide antigens that cannot be presented by major histocompatibility complex (MHC) molecules. CpG ODN is generally not effective as an adjuvant for pure polysaccharide antigens (9), but it is quite effective with protein-conjugated polysaccharides (10,11) or oligosaccharide (12). Polysaccharides are poorly antigenic in the very young and elderly populations that are most at risk of infection. However, CpG ODN are effective adjuvants for conjugated polysaccharide antigens in neonatal and very old mice (two years old) (13). Various other potential advantages have been suggested from animal studies. The more rapid appearance of antibodies allows earlier boosting while still obtaining the full boosting effect (Weeratna and Davis, unpublished results). It is possible as well to reduce the dose of antigen by 10 to 100 fold and still induce an equivalent humoral response to that with antigen alone (14,15). The possibility to use CpG in neonates or for induction of mucosal immunity is discussed in separate sections below. While most nonclinical data have been generated in mice, enhanced immunogenicity of antigens has also been demonstrated in larger animal models including guinea pigs (16), gerbils, (17) and rabbits (18). Of note, responses in rabbits are generally poor since they appear to be TLR9 deficient (unpublished data). Nonhuman primate data with CpG adjuvants include enhanced humoral and/or cell-mediated responses with HIV/SIV antigens in rhesus macaques (19–22), anthrax vaccines in macaques (16), Plasmodium falciparum CSP antigen in Aotus monkeys (23), hepatitis B surface antigen (HBsAg) in chimpanzees (24), and hepatitis C virus (HCV) antigens in baboons (25). A number of other animal species are discussed below under veterinary applications.

Mucosal Adjuvant Activity Most pathogens enter the body through one of the vast mucosal surfaces, so there is a great deal of interest in effective adjuvants for mucosal immunization. Intranasal or oral recombinant protein vaccines with CpG adjuvant are equally effective as those with CT and LT holotoxins but without the toxic effects; even doses 100 fold more than those required for optimal effect are well tolerated (26–31). Given at a mucosal route, CpG ODN still induces Th1 immune responses, both systemic and mucosal, and also drives strong mucosal IgA responses at local and distant mucosal sites (27). A particularly potent method to induce strong mucosal immunity was with systemic prime (IM) and mucosal boost (IN) or vice versa (32). CpG has also been used with oral vaccination with a Salmonella enterica serovar Typhimurium DNA delivery system vaccine against Trypanosoma cruzi (33).

Immune Deficiency: CpG Adjuvants Overcome Vaccine Hyporesponsiveness Inducing immunity in the very young is very important from the public health standpoint but extremely difficult to attain because of the immaturity and Th2 bias of the immune system at birth. Even with repeated vaccination, immune responses are generally modest in neonatal mice or humans, and thus, vaccination of babies is often postponed until the age of two to three months. In newborn mice, CpG induced earlier and stronger humoral or CTL immune responses against HBsAg (34,35), tetanus toxoid, live measles virus, and recombinant canary pox expressing measles HA (36). CpG was even able to prime antibody and CTL against HBsAg in the presence of high levels of maternal antibodies (34,35). Prior to this, successful immunization of newborn mice in the presence of maternal antibodies had been restricted to DNA vaccines. In neonates of a larger species (pigs), CpG has been used effectively to induce humoral and cellular immunity against various infectious disease antigens (37,38). Vaccine hyporesponsiveness can occur beyond the neonatal period, as, for example, during treatment with chemotherapy, with HIV infection or because of genetic factors. C57BL/6 mice immunosuppressed by cyclophosphamide experienced a two-fold increase in anti-HBsAg IgG and significant increase in IL-12 levels after coadministration of HBsAg with CpG ODN (39). SIV-infected rhesus macaques were vaccinated with commercial hepatitis B virus (HBV) vaccine containing alum as adjuvant with or without CpG ODN. Only those receiving CpG adjuvant produced detectable antibodies against HBsAg (19). Similarly, orangutans are hyporesponsive to HBsAg, likely because of genetic factors. A vaccine program to immunize orangutans in a rehabilitation center against HBV prior to their release back to the jungle gave only 15% seroprotection with two doses of a commercial vaccine, but when CpG was added, this reached 100% (40).

Other Adjuvants and Delivery Systems: Synergy with CpG The CpG adjuvant effects may be further enhanced by coadministration with other adjuvants and delivery systems. What is remarkable in these combination studies is that the strong Th1 bias is maintained or further enhanced even if the combining adjuvant has by itself a strong Th2 bias. There appears to be two primary mechanisms for such synergy. The first mechanism to explain synergy involves adjuvants and delivery systems that keep the CpG and antigen together, which likely ensures that the same APC that are presenting the antigen are also activated by the CpG. Since CpG is a relatively small molecule and antigens are often large molecules, they may distribute differently when codelivered in aqueous solutions. This seems to be especially true with subcutaneous injection, presumably because of the larger space for CpG and antigen to diffuse away from each other. Such ‘‘delivery’’ synergy has been shown with alum (41,42), calcium phosphate (43), emulsions (44), a-2-macroglobulin (45), biodegradable poly(lactide-co-glycolide) (PLG) microparticles (46,47), and gelatin microparticles (48). Another solution for maintaining the desired proximity is to conjugate the CpG directly to the antigen, as has been shown to work well for allergy-based vaccines (49), although one study showed that biodegradable microparticles gave superior

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results (50) and another showed that conjugation to the 50 -end of the ODN resulted in loss of adjuvant activity (51). The second mechanism for synergy is to combine CpG ODN with another adjuvant that works on different cells or through different pathways. Such immune enhancement synergy has been demonstrated by combining CpG with monophosphoryl lipid A (MPL), a TLR4 agonist (43,44), QS21, a saponin immune modulator of unknown mechanism of action (52), or granulocyte-macrophage colony-stimulating factor (GM-CSF) (53).

DNA Vaccines and Viral Vectors: Role of CpG Motifs DNA vaccines are plasmids naturally containing several hundred unmethylated CpG motifs, which appear to be necessary for adequate immunogenicity of the expressed antigen (54–56). It is possible to further enhance the immunogenicity of DNA vaccines by cloning in further CpG motifs (56) or coadministering it with additional noncoding vector DNA in mice (55,57–59) and primates (60,61). However, the addition of too many CpG motifs to a plasmid suppresses the humoral response, possibly because CpG-induced cytokines, such as type 1 interferons, suppress expression of antigen from the commonly used cytomegalovirus (CMV) promoter (61). It is also a delicate balance to add CpG ODN to a plasmid DNA vaccine because of the dose-dependent interference of the phosphorothioate ODN backbone with the uptake and expression of the plasmid (62,63). While gene gun delivery of DNA vaccines should avoid the cell uptake issues, immunization against an LCMV CTL epitope did not give increased levels of CTL after coating the beads with CpG ODN (64) that was delivered via gene gun. This may be due to cytokine downregulation of the plasmid promoter, or possibly the gene gun fails to deliver the CpG to the endosomal compartment where TLR9 is located. CpG ODN have also been incorporated into a live parvovirus vaccine. This combination resulted in enhanced immunogenicity and efficacy (survival from tumor challenge) in mice (65).

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hepatitis B and C where antigenic tolerance (HBV) and insufficient Th1-type T-cell responses (HBV and HCV) are thought to contribute. There are no suitable small animal models for either of these diseases, but vaccinating with recombinant HBsAg and CpG ODN can break B- and T-cell tolerance in transgenic mice that express HBsAg protein principally in the liver under the control of the endogenous HBV promoter (112). Surprisingly, the resulting immune response clears circulating HBsAg and markedly reduces HBsAg mRNA expression in the liver without causing a cytopathic effect (113). Adoptive transfer experiments showed that both IFN-g-secreting CD4 and CD8 T cells are responsible for the noncytolytic control of viral expression (114). Other chronic diseases that might benefit from treatment with a CpG-containing vaccine that would induce Th1-type cell-mediated immunity include HSV, HIV, and TB.

Cancer Vaccines The strong Th1 adjuvant effects of CpG ODN make them ideal candidates to use with tumor antigens in cancer vaccines. The antitumor adjuvant properties of CpG ODN have been shown effective in various murine tumor models with several types of vaccines including (i) tumor-derived peptide in a melanoma model (115) and cervical carcinoma model (116), (ii) tumorspecific antigen in a B-cell lymphoma model (see below), (iii) tumor lysate in a glioblastoma model, (iv) irradiated whole-cell tumor vaccine in neuroblastoma (117) and renal cell carcinoma (RENCA) models (Weeratna and Davis, unpublished results), (v) idiotype of surface IgM in the 38C13 murine B-cell lymphoma model (118), (vi) an adenoviral vector expressing tumor-specific antigen in a prostate tumor model (119), (vii) pulsed DC vaccine in the RENCA model (120), (viii) DC cocultured with irradiated tumor cells in a murine colon cancer model (121), a live parvovirus vector (65), and (ix) adoptive transfer of T cells primed in vivo and restimulated ex vivo against the tumor cells in an A20 lymphoma model (122).

Allergy Vaccines Prophylactic Infectious Disease Vaccines The greatest body of CpG adjuvant work has been carried out with infectious disease antigens, where they have been shown in animal models, predominantly mice, to be very potent for augmenting humoral and cellular responses to an extensive list of antigens of viral, bacterial, fungal, or parasitic origin (Table 1). In some cases, challenge studies have also been carried out, with enhanced immunity usually correlating with increased protection (Table 1). Interestingly, CpG ODN was able to enhance the immunogenicity of BCG vaccine and its efficacy against challenge with Mycobacterium tuberculosis, which presumably already contains immune stimulatory BCG-derived immunostimulatory CpG motifs (95). On the other hand, CpG ODN combined with M. tuberculosis culture filtrate proteins elicited enhanced IFN-g responses but did not achieve protection against challenge with M. tuberculosis (111).

Therapeutic Infectious Disease Vaccines The strong Th1-like adjuvant effects of CpG ODN along with the ability to provide T help and to overcome hypo- and nonresponsiveness to antigens make it an ideal candidate as an immune enhancer in therapeutic vaccines to treat chronic infections. Likely target indications are chronic infections of

Allergic symptoms result from Th2-type immune responses against otherwise harmless environmental antigens. Th2 cytokines such as IL-4 and IL-5 induce B cells to secrete IgE, which in turn binds to high-affinity IgE Fc receptors on the surface of mast cells and basophils. If present, allergens can then bind to such surface IgE, cross-linking the IgE Fc receptors and leading to activation and degranulation of the mast cells or basophils. These cells release a variety of preformed proinflammatory and vasoactive compounds including histamine, prostaglandins, leukotrienes, and cytokines, resulting in an immediate inflammatory response that is often followed several hours later by a secondary reaction. While antihistamines are effective for temporary control of allergic symptoms, research results have provided hope that CpG ODN, through induction of Th1-type responses, could redirect the unwanted Th2 allergic responses and provide a long-term or potentially permanent ‘‘cure’’ to allergic disease. Two basic approaches have been investigated, nonallergenspecific immune modulation (not the subject of this review) and use of CpG in allergy vaccines (123). Studies in mice with previously established allergic disease (through repeated immunization with Th2 adjuvants) have shown that vaccines containing low doses of allergens and CpG adjuvant induced T h 1-biased allergen-specific responses, reversing the established T h 2 responses and associated

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Table 1 Studies Testing Infectious Disease Vaccines with CpG Oligodeoxynucleotide as Adjuvant in Animal Models and Demonstrating Enhanced Immunogenicity and/or Challenge Outcomes Pathogen

Antigen

Immunogenicity

Challenge

Peptide

66

Hepatitis B surface antigen (VLP) VLPs Envelope, structural and nonstructural proteins gD HPV 16 L1-E7 fusion proteins HPV 16 major capsid protein Killed split VP6 JE vaccine L1, A33, B5 Receptor-binding domain of SARS-CoV spike protein SARS-CoV inactivated vaccine Envelope DIII glycoprotein Gag opg140 gp160 gp120-depleted particles Whole inactivated SIV Multiple recombinant subunit Modified vaccinia Ankara vaccine

67–69 25 70–74 75 76 31 77 78 79 80 81 82 21 (83) (84) 20, 85–89) 22 (87) 90

31 77

B. anthracis protective antigen Whole-cell sonicate Recombinant proteins (5) Whole-killed mycobacterium Escherichia coli J5 LPS þ OMP of group B Neisseria meningitidis

14, 16, 91 92, 93 94 95 96

14 92

Viral pathogens Foot and mouth disease virus (picornavirus) Hepatitis B Hepatitis C Herpes simplex virus 2 HPV Influenza Rotavirus Japanese encephalitis virus Orthopox SARS West Nile virus HIV/SIV

Smallpox

75

(87) 90

Bacterial pathogens Bacillus anthracis Helicobacter pylori Meningococcus, group B Mycobacterium tuberculosis Polymicrobial sepsis Parasites Eimeria coccidiosis Entamoeba histolytica Leishmania major Leishmania infantum (cutaneous) Leishmania donovani L. donovani L. major Malaria: Plasmodium falciparu Malaria: Plasmodium vivax Malaria: Plasmodium yoelii Trypanosoma cruzi Toxoplasma gondii

Protein antigen 2/praline-rich antigen Gal-inhibitable lectin Whole killed, recombinant Infantum acidic ribosomal P0 protein Recombinant ORFF Leishmania soluble antigen Live attenuated AMA1 Pfs25 MSP1 MSP1 ASP-2 Toxoplasma lysate antigen

17 (99) (100) (101) (102) (103) (104) (105) (106 (107)

97, 98 17

(107) (108) (109, 110)

Abbreviations: HPV, Human papillomavirus; CoV, coronavirus; gD, glycoprotein D; JE, Japanese encephalitis; SARS, severe acute respiratory syndrome; VLP, virus-like particle; LPS, lipopolysaccharide; OMP, outer membrane protein; ORFF, open reading frame; AMA1, anti-apical-membrane-antigen 1; MSP1, merozoite surface protein 1; ASP-2, actinobacteria specific protein.

asthmatic symptoms upon airway challenge (49). In theory, allergens in the vaccine could still induce anaphylactic reactions, especially during the early treatment period when strong Th2 responses would still be present. However, it was shown that the allergenicity of the vaccine could be significantly reduced by chemically conjugating the allergen to the CpG ODN (123). CpG-allergen conjugates were shown to be effective in treating mouse models of ragweed allergy, using the Amb a1 ragweed allergen (124), and dust mite allergic rhinitis, using a CpG–Dermatophagoides farinae conjugate (125). The ragweed vaccine did reach clinical testing, and this is discussed below under human experience.

VETERINARY APPLICATIONS OF CpG ODN VACCINE ADJUVANTS In many countries, more doses of vaccines are sold for veterinary than for human use. The need for an effective adjuvant is important in these situations since profit margins are low, so antigen manufacturing costs need to be minimized, and for logistical reasons, it is necessary to protect animals with as few doses as possible. Strong CpG adjuvant activity has been demonstrated with a variety of vaccines in several companion and food source species of animals. Chickens show enhanced protection against Eimeria coccidiosis (126) and Newcastle disease virus (127) with addition of

Chapter 26: TLR9 Agonists for Immune Enhancement of Vaccines

CpG ODN to vaccines. Improved humoral and cellular responses are seen in piglets by using CpG ODN adjuvant with vaccines against Toxoplasma gondii (110), porcine reproductive and respiratory syndrome (37), and pseudorabies attenuated vaccine (128). In cattle, addition of CpG ODN significantly enhanced T-cell and antibody responses to mycobacterial antigens and provided better protection against tuberculosis challenge (129). Similar enhanced immunogenicity and protection from challenge in cows were seen upon adding CpG to a bovine viral diarrhea virus vaccine (130) and a bovine respiratory syncytial virus vaccine (131,132). CpG adjuvant has also been shown to be effective in horses (133). At the lower end of the evolutionary scale, CpG ODN activate the immune systems of fish (134,135), which are commonly vaccinated within the aquaculture industry.

HUMAN EXPERIENCE WITH CpG ODN AS VACCINE ADJUVANTS Prior to testing CpG adjuvants in humans, they had been tested for their ability to activate human lymphocytes ex vivo. Since such studies gave similar results to those with mouse splenocytes (136), it was expected that they would prove to be effective adjuvants in humans. Indeed, CpG ODN adjuvants have since been administered to several thousand humans with different antigens in clinical trials and have been shown to be highly effective and well tolerated.

Infectious Disease Vaccines: Clinical Experience Immunogenicity studies have been carried out in humans with a variety of infectious disease antigens. Several of these studies have been carried out using HBsAg. In healthy volunteers, CpG ODN added to a commercial HBV vaccine containing alum adsorbed HBsAg resulted in significantly faster induction of antibodies that were of significantly higher levels (~10-fold) and avidity. Remarkably, with CpG added (0.5 mg), a single vaccine dose induced 75% seroprotection (anti-HBs  10 mIU/mL) compared with only 13% for the control vaccine (137,138). A similar study in HIVþ hyporesponders who failed previous HBV vaccination showed significantly better humoral and T-cell (lymphoproliferative) responses when CpG was added to the commercial HBV vaccine (139,140), and this difference between groups was maintained even five years later (141). In two other studies, a CpG ODN was administered with HBsAg alone (no alum). With the knowledge from animal studies that alum provides a benefit by binding the CpG and antigen together, the CpG effect would be expected to be weaker than that in the trials where an alum-based vaccine was used. Indeed, this was the case in one study, which used the same dose of CpG as had been used with the alum-based vaccines (142). However, in another trial, a higher dose of CpG was used with good results (143), and this latter vaccine, known as HEPLISAVTM [Dynavax Technologies (Berkeley, California, U.S.) and Merck & Co. Inc. (West Point, Pennsylvania, U.S.)], is now in phase III testing. Results were also disappointing when CpG was tested with a single-dose, trivalent, split influenza vaccine; in this case, antibody titers were enhanced with CpG over control only when subjects had some preexisting immunity. It is likely that the poor results can be attributed, at least in part, to the lack of alum to hold the relatively low dose of CpG and antigen together (144). CpG ODN has also been shown to be a highly effective adjuvant in healthy volunteers with other antigens when alum

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was included. Adding CpG ODN to BioThrax1 [anthrax vaccine adsorbed (AVA)], a commercial Bacillus anthracis vaccine (that contains protective antigen along with other B. anthracis proteins), resulted in significantly higher peak antibody response and a three-week shorter time to attainment of a seroprotective antibody compared with AVA alone (91). In a phase I study of a P. falciparum malaria vaccine (AMA1-C1) with CpG ODN plus alum as adjuvants, an 8- to 10-fold increase in anti-AMA1 titers was observed versus alum alone (145).

Cancer Vaccines: Clinical Experience CpG ODN has been utilized as an adjuvant with a number of tumor antigens in oncology patients. In these cases, measurement of T-cell responses is thought to be the best measure for potential efficacy. Strong antigen-specific T cells have been induced in melanoma patients by adding CpG to NY-ESO peptide (146,147).

Allergy Vaccines: Clinical Experience As discussed above, the concept of CpG-containing allergy vaccines is to redirect previously existing symptom-causing Th2 responses against an allergen into nonsymptomatic Th1-type responses. TOLAMBATM (Dynavax) is a vaccine comprising the Amb a1 allergen of ragweed conjugated to CpG. In phase I and II clinical trials in subjects with ragweed allergy, the TOLAMBA vaccine was shown to be well tolerated and to induce Amb a1–specific IgG but not IgE (49). However, a phase III trial failed to meet its primary end points, and the development program was stopped. The reason for the failed trial is unclear, although there were issues of placebo-treated subjects having lower than expected symptoms during the following ragweed season. Therefore, it is unclear whether this approach will one day be used for clinical benefit. There are those who believe that allergen-specific immunotherapy (i.e., allergy vaccines) will require a long treatment time with a large number of doses (148). If this is true, allergy vaccines will offer fewer advantages over classical desensitization therapy than originally hoped. Nevertheless, the possibility to reduce the risk of anaphylactic shock is not insignificant (124).

SAFETY OF CpG ODN In the numerous animal models reviewed in the previous sections, CpG ODN has proven to be not only potent but also well tolerated across a wide range of doses. In comparative studies in animals, CpG has been shown to be less reactogenic in mice than other adjuvants (44). In marmosets, incomplete Freund’s adjuvant with a Plasmodium vivax vaccine caused ulceration at the injection site, whereas CpG did not (106). In humans, CpG ODN vaccine adjuvants have also been shown in several clinical trials with different antigens to be well tolerated when delivered by intramuscular or subcutaneous injection (138–147,149). Effects of the ODN backbone per se were not expected to pose problems, since chemically similar molecules had been given in doses of higher orders of magnitude and much more frequently in antisense trials. Prior to testing began in humans, the greatest perceived risk with CpG ODN was that its strong Th1 immune effects might induce autoimmunity, especially against DNA. Natural environmental exposures to CpG ODN in the form of infections are quite frequent and have not been shown to lead to an increased risk of autoimmune disease in humans, although

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viral infections frequently induce anti-ssDNA antibodies and may even induce anti-dsDNA antibodies (150). The risk for developing lupus or other autoimmune diseases is generally not increased among patients with chronic infections who are presumably chronically exposed to high concentrations of CpG DNA. Moreover, hundreds of humans have received antisense ODN, which are made with the same synthetic backbone as CpG ODN and many of which happen to have immune stimulatory CpG motifs, with no reports of association with autoimmunity despite the very high doses administered (151). Thus, any added risk from use of low-dose CpG ODN for vaccine adjuvant purposes is thought to be small. Several thousand humans have now received CpG ODN as a vaccine adjuvant in clinical trials. Among these trials, there has been a single report of Wegener’s granulomatosis, an autoimmune vascular condition, in a phase III trial with HEPLISAV, a prophylactic vaccine against hepatitis B that contains a CpG ODN (152). This trial is currently on clinical hold pending investigation of this case. Potential safety concerns have been addressed in a wide range of studies, and this overall experience has been reviewed by Krieg (153). Collectively, the data indicate that CpG DNA does not generally abrogate B- or T-cell tolerance, or induce autoantibody production or autoimmune disease even in genetically predisposed individuals.

SUMMARY CpG ODN is a promising Th1-type immune stimulant that promises to be highly effective in vaccines against infectious diseases, cancer and allergies. For infectious diseases, it may allow the induction of protective immune responses with fewer and lower doses of antigen, even in neonates and hyporesponders. Most important is the possibility to produce therapeutic vaccines to treat chronic viral infections and cancer through induction of potent T-cell responses or to treat asthma by redirecting preexisting Th2 responses to Th1. CpG ODN have been shown to be highly effective for augmenting immune responses against various antigens in numerous human and veterinary clinical studies. While the ability to augment humoral responses has been clearly demonstrated, additional clinical studies with appropriate antigens are required to determine the true potential of CpG ODN in therapeutic vaccines.

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65. Raykov Z, Grekova S, Leuchs B, et al. Arming parvoviruses with CpG motifs to improve their oncosuppressive capacity. Int J Cancer 2008; 122(12):2880–2884. 66. Beignon AS, Brown F, Eftekhari P, et al. A peptide vaccine administered transcutaneously together with cholera toxin elicits potent neutralising anti-FMDV antibody responses. Vet Immunol Immunopathol 2005; 104(3–4):273–280. 67. Davis HL, Weeratna R, Waldschmidt TJ, et al. CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. J Immunol 1998; 160(2): 870–876. Erratum in: J Immunol 1999; 162:3103. 68. Schirmbeck R, Melber K, Reimann J. Adjuvants that enhance priming of cytotoxic T cells to a Kb-restricted epitope processed from exogenous but not endogenous hepatitis B surface antigen. Int Immunol 1999; 11(7):1093–1102. 69. Wild J, Grusby MJ, Schirmbeck R, et al. Priming MHC-I-restricted cytotoxic T lymphocyte responses to exogenous hepatitis B surface antigen is CD4þ T cell dependent. J Immunol 1999; 163(4): 1880–1887. 70. Ma X, Forns X, Gutierrez R, et al. DNA-based vaccination against hepatitis C virus (HCV): effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice. Vaccine 2002; 20(27–28):3263–3271. 71. Jiao X, Wang RY, Qiu Q, et al. Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes. J Gen Virol 2004; 85(pt 6): 1545–1553. 72. Yu H, Babiuk LA, van Drunen Littel-van den Hurk S. Priming with CpG-enriched plasmid and boosting with protein formulated with CpG oligodeoxynucleotides and Quil A induces strong cellular and humoral immune responses to hepatitis C virus NS3. J Gen Virol 2004; 85(pt 6):1533–1543. 73. Sugauchi F, Wang RY, Qiu Q, et al. Vigorous hepatitis C virusspecific CD4þ and CD8þ T cell responses induced by protein immunization in the presence of Montanide ISA720 plus synthetic oligodeoxynucleotides containing immunostimulatory cytosine-guanine dinucleotide motifs. J Infect Dis 2006; 193(4): 563–572. 74. Vajdy M, Selby M, Medina-Selby A, et al. Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses. J Gen Virol 2006; 87 (pt 8):2253–2262. 75. Gallichan WS, Woolstencroft RN, Guarasci T, et al. Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract. J Immunol 2001; 166(5):3451–3457. 76. Tho¨nes N, Mu¨ller M. Oral immunization with different assembly forms of the HPV 16 major capsid protein L1 induces neutralizing antibodies and cytotoxic T-lymphocytes. Virology 2007; 369:375–388. 77. Choi AH, McNeal MM, Flint JA, et al. The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant. Vaccine 2002; 20(13–14):1733–1740. 78. Sidhapuriwala JN, Sivalingam SP, Lu J, et al. Immunomodulation of Japanese encephalitis vaccine through CpG oligodeoxynucleotides in mice. Scand J Immunol 2006; 64(4):370–375. 79. Fogg CN, Americo JL, Lustig S, et al. Adjuvant-enhanced antibody responses to recombinant proteins correlates with protection of mice and monkeys to orthopoxvirus challenges. Vaccine 2007; 25(15):2787–2799. 80. Zakhartchouk AN, Sharon C, Satkunarajah M, et al. Immunogenicity of a receptor-binding domain of SARS coronavirus spike protein in mice: implications for a subunit vaccine. Vaccine 2007; 25(1):136–143. 81. Zhang CH, Lu JH, Wang YF, et al. Immune responses in Balb/c mice induced by a candidate SARS-CoV inactivated vaccine prepared from F69 strain. Vaccine 2005; 23(24):3196–3201.

82. Chu JH, Chiang CC, Ng ML. Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection. J Immunol 2007; 178(5):2699–2705. 83. Rao M, Matyas GR, VanCott TC, et al. Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein. Immunol Cell Biol 2004; 82(5):523–530. 84. Deml L, Schirmbeck R, Reimann J, et al. Immunostimulatory CpG motifs trigger a T helper-1 immune response to human immunodeficiency virus type-1 (HIV-1) gp 160 envelope proteins. Clin Chem Lab Med 1999; 37(3):199–204. 85. Moss RB, Diveley J, Jensen F, et al. In vitro immune function after vaccination with an inactivated, gp120-depleted HIV-1 antigen with immunostimulatory oligodeoxynucleotides. Vaccine 2000; 18(11–12):1081–1087. 86. Ayash-Rashkovsky M, Borkow G, Davis HL, et al. Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection. FASEB J 2005; 19:1152–1154. 87. Xiao Y, Aldaz-Carroll L, Ortiz AM, et al. A protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost. Vaccine 2007; 25(7):1214–1224. 88. Azizi A, Ghorbani M, Kryworuchko M, et al. Potency of cellmediated immune responses to different combined HIV-1 immunogens in a humanized murine model. Hum Vaccin 2005; 1(4):170–176. 89. Trabattoni D, Clivio A, Bray DH, et al. Immunization with gp120depleted whole killed HIV immunogen and a second-generation CpG DNA elicits strong HIV-specific responses in mice. Vaccine 2006; 24(9):1470–1477. 90. Belyakov IM, Isakov D, Zhu Q, et al. Enhancement of CD8þ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective efficacy of a modified vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-deficient host. J Immunol 2006; 177(9):6336–6343. 91. Rynkiewicz D, Rathkopf M, Ransom J, et al. Marked enhancement of antibody response to anthrax vaccine absorbed with CPG 7909 in healthy volunteers. 45th Interscience Conference on Antimicrobal Agents and Chemotherapy. Washington, DC, 2005. 92. Shi T, Liu WZ, Gao F, et al. Intranasal CpG-oligodeoxynucleotide is a potent adjuvant of vaccine against helicobacter pylori, and T helper 1 type response and interferon-gamma correlate with the protection. Helicobacter 2005; 10(1):71–79. 93. Hatzifoti C, Bajaj-Elliott M, Dorrell N, et al. A plasmid immunization construct encoding urease B of Helicobacter pylori induces an antigen-specific antibody response and upregulates the expression of beta-defensins and IL-10 in the stomachs of immunized mice. Vaccine 2004; 22(20):2651–2659. 94. Giuliani MM, Adu-Bobie J, Comanducci M, et al. A universal vaccine for serogroup B meningococcus. Proc Natl Acad Sci U S A 2006; 103(29):10834–10839. 95. Freidag BL, Melton GB, Collins F, et al. CpG oligodeoxynucleotides and interleukin-12 improve the efficacy of Mycobacterium bovis BCG vaccination in mice challenged with M. tuberculosis. Infect Immun 2000; 68(5):2948–2953. 96. Opal SM, Palardy JE, Chen WH, et al. Active immunization with a detoxified endotoxin vaccine protects against lethal polymicrobial sepsis: its use with CpG adjuvant and potential mechanisms. J Infect Dis 2005; 192(12):2074–2080. 97. Dalloul RA, Lillehoj HS, Klinman DM, et al. In ovo administration of CpG oligodeoxynucleotides and the recombinant microneme protein MIC2 protects against Eimeria infections. Vaccine 2005; 23(24):3108–3113. 98. Kirkland TN, Raz E, Datta SK. Molecular and cellular mechanisms of protective immunity to coccidioidomycosis. Vaccine 2006; 24(4):495–500.

Chapter 26: TLR9 Agonists for Immune Enhancement of Vaccines 99. Rhee EG, Mendez S, Shah JA, et al. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4þ and CD8þ T cell responses and protection against leishmania major infection. J Exp Med 2002; 195(12):1565–1573. 100. Iborra S, Carrion J, Anderson C, et al. Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice. Infect Immun 2005; 73(9):5842–5852. 101. Tewary P, Sukumaran B, Saxena S, et al. Immunostimulatory oligodeoxynucleotides are potent enhancers of protective immunity in mice immunized with recombinant ORFF leishmanial antigen. Vaccine 2004; 22(23–24):3053–3060. 102. Tewary P, Pandya J, Mehta J, et al. Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection. FEMS Immunol Med Microbiol 2004; 42(2): 241–248. 103. Wu W, Weigand L, Belkaid Y, et al. Immunomodulatory effects associated with a live vaccine against Leishmania major containing CpG oligodeoxynucleotides. Eur J Immunol 2006; 36(12): 3238–3247. 104. Mullen GE, Giersing BK, Ajose-Popoola O, et al. Enhancement of functional antibody responses to AMA1-C1/Alhydrogel, a Plasmodium falciparum malaria vaccine, with CpG oligodeoxynucleotide. Vaccine 2006; 24(14):2497–2505. 105. Qian F, Rausch KM, Muratova O, et al. Addition of CpG ODN to recombinant Pseudomonas aeruginosa ExoProtein A conjugates of AMA1 and Pfs25 greatly increases the number of responders. Vaccine 2008; 26(20):2521–2527. 106. Rosa DS, Iwai LK, Tzelepis F, et al. Immunogenicity of a recombinant protein containing the Plasmodium vivax vaccine candidate MSP1(19) and two human CD4þ T-cell epitopes administered to non-human primates (Callithrix jacchus jacchus). Microbes Infect 2006; 8(8):2130–2137. 107. Jeamwattanalert P, Mahakunkijcharoen Y, Kittigul L, et al. Longlasting protective immune response to the 19-kilodalton carboxyterminal fragment of Plasmodium yoelii merozoite surface protein 1 in mice. Clin Vaccine Immunol 2007; 14(4):342–347. 108. Araujo AF, de Alencar BC, Vasconcelos JR, et al. CD8þ-T-celldependent control of Trypanosoma cruzi infection in a highly susceptible mouse strain after immunization with recombinant proteins based on amastigote surface protein 2. Infect Immun 2005; 73(9):6017–6025. 109. El Malky M, Shaohong L, Kumagai T, et al. Protective effect of vaccination with Toxoplasma lysate antigen and CpG as an adjuvant against Toxoplasma gondii in susceptible C57BL/6 mice. Microbiol Immunol 2005; 49(7):639–646. 110. Kringel H, Dubey JP, Beshah E, et al. CpG-oligodeoxynucleotides enhance porcine immunity to Toxoplasma gondii. Vet Parasitol 2004; 123(1–2):55–66. 111. Fonseca DM, Silva CL, Paula MO, et al. Increased levels of interferon-gamma primed by culture filtrate proteins antigen and CpG-ODN immunization do not confer significant protection against Mycobacterium tuberculosis infection. Immunology 2007; 121(4):508–517. 112. Malanchere-Bres E, Payette PJ, Mancini M, et al. CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice. J Virol 2001; 75(14): 6482–6491. 113. Mancini M, Davis H, Tiollais P, et al. DNA-based immunization against the envelope proteins of the hepatitis B virus. J Biotechnol 1996; 44(1–3):47–57. 114. Mancini M, Hadchouel M, Tiollais P, et al. Regulation of hepatitis B virus mRNA expression in a hepatitis B surface antigen transgenic mouse model by IFN-g-secreting T cells after DNAbased immunization. J Immunol 1998; 161(10):5564–5570.

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27 Use of Genetically or Chemically Detoxified Mutants of Cholera and Escherichia coli Heat-Labile Enterotoxins as Mucosal Adjuvants Lucy C. Freytag and John D. Clements Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, Louisiana, U.S.A.

Dubravka Grdic Eliasson and Nils Lycke Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden

INTRODUCTION Over the last 10 to 15 years, a great deal of effort has been directed toward replacement of existing whole-cell or formalininactivated vaccines with subunit vaccines that may be safer and more effective than existing vaccines. Still other efforts are directed at developing alternatives to traditional vaccine delivery, including mucosal and transcutaneous delivery. It is generally agreed that the latter routes offer a number of potential advantages over traditional vaccines, including (i) elimination of needles and the risk of transferring disease by contamination, (ii) the potential to confer mucosal as well as systemic immunity, (iii) increased stability and perhaps also less of a need for an intact cold-chain, and (iv) that administration would not require trained health care specialists. However, it has become increasingly clear that a major limiting factor for the development of mucosal and transcutaneous vaccines is the availability of safe and, above all, effective adjuvants. Because of the requirement for adjuvants for many nonliving vaccines, there is growing interest in adjuvant development. Despite this, we must conclude that little information is available as to what adjuvants, in fact, do in vivo. Although the literature is extensive on adjuvants and their function, much of our mechanistic knowledge stems from in vitro studies using freshly isolated cells, cell lines, or ex vivo generated cells, such as bone marrow–derived dendritic cells (DC) (1). We believe that the main target for adjuvants is the innate immune system and especially antigen-presenting cells (APC). As these cells are exposed to adjuvant they upregulate costimulatory molecules and release predominantly pro-inflammatory cytokines and chemokines, which will affect priming of T and B cells at the inductive sites of an immune response (2). However, the consequences that this interaction may have for antigen processing, cell migration, and differentiation in vivo awaits more detailed investigation (3,4). With the introduction of multi-photon microscopy and other real-time assessments, cellular movement and function due to adjuvant administration are soon likely to generate a rapidly growing body of evidence (5–7). This is a much warranted development for future vaccine adjuvant design and formulation.

In the present chapter, we discuss aspects of adjuvanticity, focusing on mutants or engineered derivatives of cholera toxin (CT) and Escherichia coli heat-labile toxin (LT). We will summarize what is known about their mechanism of action and discuss their clinical applicability for future mucosal or transcutaneous vaccines.

MUCOSAL IMMUNIZATION The first productive interaction between most infectious agents and the human host is with mucosal surfaces, specifically, the nasal, oropharyngeal, respiratory, genitourinary, and gastrointestinal mucosa. These vast surface areas, extending over 400 m2, are the barriers against invading pathogens, but also the sites for host interactions with the commensal flora and food antigens. In keeping with this, mucosal membranes are strictly regulated immune environments, where nonresponsiveness, that is, tolerance, can be found alongside with the ability to raise substantial protective immunity (8). It is still poorly understood how this dichotomy is maintained and what constitutes the regulatory machinery responsible for tolerance and protective immunity. However, harnessing the latter, secretory immunoglobulin A (sIgA), is the first line of defense against invading pathogens, but also a link in the mutualism that is established between the host and the commensal flora in the gut (9–11). Specific IgA antibodies may block attachment of bacteria and viruses, neutralize bacterial toxins, and even inactivate invading viruses inside of epithelial cells (ECs) (11,12). In addition the mucosal immune system is an integrated system that allows immunizations at one mucosal site to stimulate production of specific IgA also at distant mucosal sites. For example, intranasal immunization stimulates strong mucosal immunity in the respiratory tract as well as in the genital tract. However, such integration is not always a rule as rectal and oral immunizations stimulate IgA antibody production confined primarily to the gastrointestinal tract (13). Of note, mucosal immunization can be an effective means of inducing not only sIgA but also systemic IgG antibodies and cell-mediated immunity. On the contrary, parenteral immunizations most often fail to stimulate mucosal IgA immunity (14).

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GENERAL PRINCIPLES FOR ADJUVANT ACTIONS Adjuvants may use many different mechanisms to exert an augmenting effect on immune responses: From establishing an antigen-depot in the tissue, to direct or indirect immunomodulation and antigen-targeting effects. For example, nonliving adjuvants can be formulations of lipid or gel (alum) to create a depot effect of the vaccine following injection. Also, nonliving adjuvants can be both delivery systems, such as liposomes and polylactide/polyglycolide (PLG) microspheres, or modulators such as muramyl dipeptide (MDP) and monophosphoryl lipid A (MPL) (15). In principle, adjuvants may exploit three types of modulating effects on the innate immune response, which will impact on the adaptive immune response and promote improved immunogenicity that can eventually convey protection against infection. First, designated receptors for microbial recognition could be used. The pattern recognition receptors (PRRs) bind microbial products, such as endotoxin or other microbial membrane products or bacterial or viral DNA and RNA, respectively (16). Secondly, other non-signaling cell membrane molecules, such as gangliosides, may be targeted by adjuvant active immunomodulators. In this latter category we find the bacterial enteroxins elaborated by Vibrio cholerae and E. coli, that is, CT and the closely related heat-LT, respectively. A third category of immunomodulators uses endogenous elements that activate innate immunity, such as cytokines, complement fragments, or targeted antibodies (2). One family of PRRs is the Toll-like receptors (TLR) that bind LPS, flagellin, HSP60, CpG DNA, dsRNA or peptidoglycans, all with unique and distinct receptors (16–18). The family of TLR is growing and currently it encompasses 10 members in humans and 12 in mice (19). The nucleotide-binding oligomerization domain (NOD) proteins are another example of receptors that can sense microbial products (20–22). The TLRs are distinctly located to the membranes or intracellular compartments, while NODs are intracellular PRRs. Both can be found in many types of cells, among them DC, macrophages, and B cells, which are also the most important cells for antigen presentation to naive T cells and stimulation of an adaptive immune response (23). In the case of DC, the binding to PRRs will cause the migration to regional lymph nodes and the maturation of the DC into effective APC, expressing co-stimulatory molecules required for optimal T-cell priming (24). Thus, most adjuvants are ligands for PRR’s and binding results in signaling that eventuate in activation of the transcription factor nuclear factor-kappaB (NF-kB). The NF-kB translocates to the nucleus where it may stimulate expression of a whole range of genes, many of which drive inflammation (25). However, we also know of adjuvants that do not appear to involve PRRs and subsequently should be expected to be less dependent on the NF-kB pathway for their function.

ADP-RIBOSYLATING ENTEROTOXINS ARE POTENT ADJUVANTS To this category of adjuvants, which most likely affect innate immunity through other receptors than the PRRs, we count the holotoxins CT and LT (26–30). The adjuvant effect of CT was first demonstrated by Elson and Ealding in 1984 (31) and later for LT by Dickinson and Clements (30). In these studies, it was shown that CT and LT substantially increased the serum antibody response to a coadministered antigen given orally.

This effect was determined to be a function of the enzymatically active A-subunit of the toxin, because recombinant B-subunit alone was unable to augment the response. Since then the literature has grown and several groups have reported adjuvant effects also from the B-subunits, although it would be fair to say that recombinant CTB and LTB are substantially weaker adjuvants than the respective holotoxin. In fact, CTB and LTB have been used most frequently to potentiate mucosal tolerance rather than as enhancers of immune responses (32–34). Only a few studies have addressed whether adjuvanticity of the holotoxins could be dependent on TLR signaling. However, Kawamura et al. demonstrated that the effect of CT was independent of TLR4, but resulted in a GM1-dependent nuclear translocation of NF-kB, suggesting that NF-kB may be involved in the adjuvant function, although through a mechanism unrelated to the TLR (35). These holotoxins are known to be AB5-complexes and carry an A1-subunit that is an ADP-ribosylating enzyme and five B-subunits that bind distinct ganglioside receptors present on most nucleated mammalian cells. The mechanism of adjuvanticity of the ADP-ribosylating enterotoxins has long been the subject of considerable debate. Most investigators agree, though, that both elements, binding and enzymatic activity, can contribute to the immunomodulation. There is ample evidence in the literature to support this notion and in particular studies with LT and mutants thereof have documented this point (36). However, the ADP-ribosyltransferase activity appears to be key to an optimal immunoenhancing effect, as elegantly shown by Rappuoli and coworkers using mutants of LT with varying degrees of reduced enzymatic activity (37). Moreover, these molecules can exert their adjuvant function by interacting with a variety of cell types, including ECs, DCs, macrophages, B and T lymphocytes. Alternatively, CT impacts on a series of events, involving multiple steps and several cell types, to exert adjuvant function in vivo. The use of real-time multiphoton imaging could help in defining these steps (6). Undoubtedly, DCs are very important and several studies have documented their critical role for the adjuvant effect, be it enhanced expression of CD80 or 86, or production of a key cytokine, such as interleukin (IL)-6 or IL-1, as assessed in vitro using cell lines or freshly isolated APCs (38–40). In mice, though, CT injected parenterally accumulates in the marginal zone of the spleen and triggers splenic DCs to upregulate CD86, but not CD80 (39). Thus, our in vitro and in vivo findings appear to be conflicting, and it is unclear what is the key element of adjuvanticity stimulated by the holotoxins. Whether the effect of CT on DCs is direct or indirect is also important as a majority of DC in the spleen upregulate CD86, which would argue for an indirect rather than direct effect on the DC (39). Whether CT adjuvanticity involves upregulation of CD86 is difficult to test, though, because CD86–/– mice have been found to poorly respond with mucosal IgA antibodies to intranasal CTB-adjuvanted immunizations (41). Hence, it is difficult to dissociate the critical role of DCs as APC in priming of naive T cells from the adjuvant effect of CT and CTB potentially acting through CD86 on DCs as APC.

MECHANISM OF ADP-RIBOSYLATION As aforementioned, the A-subunit is the enzymatically active moiety and consists of two chains, A1 and A2, joined by a proteolytically sensitive peptide (Arg192) subtended by a

Chapter 27: Use of Detoxified CT and LT as Mucosal Adjuvants

disulfide loop. Like other A-B bacterial toxins, LT and CT require nicking and disulfide reduction to be fully biologically active. When LT or CT first encounter a mammalian cell, they bind to the surface through interaction of the B-subunit pentamer. The principle receptor for both LT-B and CT-B is GM1-ganglioside, a glycosphingolipid found ubiquitously on the surface of mammalian cells (42). A principal effect of the B-subunit interaction with mammalian cells is the stable crosslinking of GM1 at the cell surface. The A2 peptide of LT and CT facilitates association of A1 with the B-pentamer and may help direct retrograde transport of these molecules through the Golgi cisternae to the ER (43). Once in the ER, the A1 chain must be transported across the membrane into the cytosol. Evidence suggests that A1 may be transported through the Sec61p channel (44). Tsai et al. (45) have recently demonstrated that protein disulfide isomerase (PDI) in the lumen of the ER functions to disassemble and unfold CT once its A-chain has been proteolytically cleaved at Arg192. The unfolding of A1 by this redox-driven chaperone could facilitate the transport of A1 into the cytosol from the ER. The in vitro enzymatic activity of A1 is highly dependent on a family of protein cofactors, termed ‘‘ADP-ribosylating factors’’ (ARF) that belongs to the superfamily of regulatory GTPases. Recent studies have identified sites of interaction between LT-A1 and ARF3 (46). The in vivo contributions of ARF to the enzymatic activities of LT and CT are less well established. If ARF does interact with A1 in vivo, the most likely place for that to happen is in the cytosol after A1 has been secreted from the ER apart from B and A2. However, ARF could also play a role in the targeting of toxin containing endosomes through the trans-Golgi network. The A1-ARF interaction lowers the binding constants for the A1 substrates, NADþ and the a-subunit of one member of the heterotrimeric GTP-binding protein family (Gsa). When A1 transfers the ADP-ribose moiety from NADþ to Gsa while GTP is bound, Gsa is incapable of hydrolyzing GTP through its normal interaction with GTPase-activating protein (GAP). Consequently, Gsa targets, such as adenylyl cyclase, are irreversibly activated, leading to an elevation in intracellular cyclic AMP (cAMP). Increased levels of cAMP activate protein kinase A (PKA), which phosphorylates and opens the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel. Cl– efflux results in the concomitant osmotic movement of water into the gut lumen and characteristic profuse watery diarrhea.

STRATEGIES OF DETOXIFICATION OF CT AND LT Because gangliosides reside in the cell membrane of all nucleated cells, the binding of CT and LT is promiscuous and hence, these molecules can affect virtually all cells in the human body. This includes ECs and nerve cells, which render these toxin adjuvants, or derivatives thereof, unattractive for clinical use (47,48). Indeed, a commercial intranasal Flu-vaccine with LT as the adjuvant revealed increased incidence of cases with Bell’s palsy in vaccinated subjects, and led to the withdrawal of the vaccine from the market (49,50). Also, accumulation of GM1binding holotoxin and elicitation of pro-inflammatory responses in the murine brain were recorded following intranasal delivery of CT (47,51). This has prompted several groups to develop mutant toxins or derivatives thereof that have reduced toxicity while retaining strong adjuvant function. Essentially two different strategies have been employed in the

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search for better, less toxic adjuvants on the basis of this approach (Fig. 1). The first strategy was pioneered by Dickinson and Clements and Burnette et al., who independently developed mutant LT holotoxins that entirely lacked or had substantially reduced enzymatic activity (30,52). In attempts to dissociate the enterotoxic effects of CT and LT from their adjuvant activity, mutations in both the active site and the protease site of these two molecules were examined. These proved to be very successful strategies and many preclinical as well as some early clinical trials have demonstrated promising results (53,54). The single amino acid mutations most frequently used are LT-R192G, LT-A72R, LT-S63K, or CTE112K (37,54–76). The latter three are located in the A1-subunit and although dramatically less toxic were found to retain substantial adjuvant function (77). The former was engineered by Dickinson and Clements, who constructed a mutant of LT containing a single amino acid substitution altering the site of proteolytic cleavage within the disulfide subtended region joining A1 and A2: Arg at position 192 to Gly (30). Although this LT(R192G) mutant is resistant to trypsin activation, has reduced activity on mouse Y-1 adrenal tumor cells, reduced ability to induce accumulation of cAMP in cultured cells, reduced enterotoxicity in experimental animals and humans when compared with native LT, and is devoid of detectable in vitro ADP-ribosyltransferase activity, it retains the ability to function as an adjuvant when administered intranasally, orally, rectally, or transcutaneously (78–99). The attenuation of LT by removing trypsin sensitivity may be explained by the observations of Tsai et al. (45). These investigators demonstrated that unfolding of the A-subunit in the ER by PDI is dependent on proteolytic cleavage of A1 from A2. Thus, a LT-A(R192G) mutant could not be unfolded by PDI and A1 could not, therefore, be transported from the ER to the cytosol where it would be activated for ARF binding, enabling the ADPribosylation of Gsa. While that observation helps explain the attenuation of LT(R192G), it does not explain why LT(R192G) retains undiminished adjuvant activity when compared with native LT. Even though the site-directed mutagenesis strategy has been successful, it must be noted that because of the B-subunits, these mutants still carry a risk of accumulating in the central nervous tissues following intranasal immunizations (47,50). Perhaps a second generation of double mutant CT-adjuvants, such as dmCTE112K/KDEV, could avoid accumulating in the central nervous tissues following intranasal administration (66). Nevertheless, other experimental data indicate that the adjuvant dose of the holotoxins is related to the toxic dose. Therefore, one can anticipate that it will be difficult to separate adjuvanticity from toxicity in a human vaccine based on mutants of the holotoxins, if these are exploited for intranasal vaccines. However, for other routes, such as rectal, sublingual, or transcutaneous vaccination, it appears that no increased risk of side effects has been noted (32,100,101). For example, the skin-patch vaccine delivery with LT as antigen and adjuvant has enabled technical progress of a patch-vaccine product and a preliminary field efficacy trial of the LT-based travelers’ diarrhea vaccine has also validated this approach. Furthermore, combining holotoxins not only with protein antigens but also with DNA vaccines for transcutaneaous delivery could boost immunogenicity as demonstrated with LT plus an influenza DNA vaccine (102). Moreover, transcutaneous immunizations using combinations of two adjuvants such as those with CT and

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Figure 1 This schematic drawing depicts the different strategies used to detoxify the CT and LT holotoxins. The site-directed mutagenesis approach was used to introduce changes in the encoding region of the active site in A1 (LTS63K, LTA72R, CTE112K) or the protease site between A1 and A2 (LTR192G). Alternatively, elongation to truncate the A1-subunit was also tested. A third strategy was to keep the full enzymatic activity, but replace the A2- and B-subunits with a dimer of Staphylococcus aureus protein A, CTA1-DD, so that the target for cell attachment is limited to B lymphocytes rather than the wide array of cells that express GM1 ganglioside. Abbreviations: CT, cholera toxin; LT, labile toxin.

CpG ODN have proven more effective than using either adjuvant alone for stimulating anti-chlamydial immunity in the genital tract (103). Thus, the synergistic effect may result in better protection against infection compared with either adjuvant used alone following transcutaneous immunizations. An alternative approach to keeping the AB5-complex intact was developed by Lycke and coworkers (104). These investigators eliminated the B-subunits and replaced these elements with a cell-targeting element, the D-fragment from Staphylococcus aureus protein A (104,105). Hereby, the full enzymatic activity of CTA1 could be exploited in a molecule that was now nontoxic and had lost its promiscuous binding, but which retained and exerted comparable adjuvant function to that of CT holotoxin (104,106). This was also the first study to unequivocally show that the A1-subunit of the holotoxin hosted adjuvant effects, independent of the B-subunits.

CTA1-DD: KEEPING THE FULL ENZYMATIC ACTIVITY To circumvent the toxicity problem, the enzymatic activity of CTA1 was retargeted, away from the GM1-ganglioside receptor binding, by placing the gene encoding CTB in frame with a gene that encoded the D-fragment in a novel plasmid. The gene fusion protein that was produced failed to bind GM1-ganglioside but could bind to the Ig-receptor on B cells (104,107). The CTA1-DD adjuvant was found to be completely nontoxic

(104,105). Mice and monkeys were given doses of more than 500 mg of CTA1-DD without any apparent side effects or signs of reactogenicity, while similar doses of CT are known to be lethal. Thus, CTA1-DD appears to be a safe and nontoxic mucosal vaccine adjuvant, although it carries an equal ADPribosylating ability compared with CT holotoxin. Interestingly, its ability to induce cAMP in target cells is more than 10,000fold lower than that of CT, arguing against the contention that cAMP-increases are critical elements in the adjuvant function (108). CTA1-DD was shown to be as potent as the CT holotoxin in augmenting immune responses after systemic as well as mucosal immunizations (104,106,108). The adjuvant effect of CTA1-DD was dependent on an intact CTA1 enzymatic activity, as well as the Ig-binding ability of the DD-dimer (106). Mutations that obliterated the enzymatic activity (R7K or E112K) or the DD-binding ability dramatically reduced the adjuvant effect of CTA1-DD. Importantly, CTA1-DD adjuvant did not bind or accumulate in the nervous tissues after intranasal administration (109). No signs of local inflammation in the nasal mucosa or cellular deposition of inflammatory cells in the lamina propria or the organized nasal-associated lymphoid tissues (NALT) was found after intranasal administration. This finding also corroborated the observation of absence of a local edema in the footpad after injection, whereas CT elicits a substantial edema when injected into the footpads of mice (105). Thus, the CTA1DD is a highly promising mucosal and systemic adjuvant that does not appear to cause local inflammation and is safe to use

Chapter 27: Use of Detoxified CT and LT as Mucosal Adjuvants

in intranasal vaccines. The CTA1-DD adjuvant is currently being tested in the context of several candidate vaccines as part of ongoing international collaborations. In particular, promising results have been obtained with candidate mucosal vaccines against Influenza A virus, HIV Env gp 140, Helicobacter pylori, Chlamydia trachomatis, and rotavirus (110–112). Recently, we reported strong anti-influenza protective immunity in a mouse model following intranasal immunization with CTA1DD and an M2e-based vaccine formulation. The adjuvant effect was significant as it enhanced the survival and reduced morbidity to a challenge infection in immunized compared with mice immunized with influenza M2e-antigen alone (113). A further development of a candidate mucosal anti-influenza pandemic vaccine was achieved when a gene fusion protein containing the M2e-epitope in the adjuvant construct, CTA13M2e-DD, proved to be even more effective at stimulating antiinfluenza A protective immunity after intranasal immunizations (114). These findings demonstrated that a single-gene fusion protein containing immunomodulator (CTA1), vaccine antigen (M2e), and a cell-targeting element (DD) can function as a complete vaccine to stimulate protective immunity against infection.

MECHANISMS OF ADJUVANTICITY The molecular interactions and the cellular subsets through which LT and CT mediate their adjuvant properties are not completely understood, although significant efforts have been expended to resolve this important question (27–30,99,115–131). Clearly the adjuvant effect involves the modulation of the innate immune system, but it is less well understood which APCs are functionally targeted by CT and LT, in vivo. All nucleated cells, including all professional APC, can bind the toxin via the GM1-ganglioside receptor present in the cell membrane. Previous reports have documented both a proinflammatory and an anti-inflammatory effect of CT. From several studies, including our own work, CT and LT exposure of APC has an augmenting effect on IL-1 and IL-6 production, whereas in other studies a downregulating effect on IL-12, TNF-a and NO, and a promoting effect on IL-10 production have been reported (132–134). Taken together this would indicate both a pro- and anti-inflammatory effect. In fact, investigators have used the CT-adjuvant to generate Th1 cells, but most reports have shown a bias for Th2 cells, and recently also IL-10 producing regulatory Tr-1 cells (116,129,135). In this context it is interesting to note that another ADP-ribosylating toxin, pertussis toxin (PT), has been reported to reduce Tregactivity, while CT was found to specifically stimulate Tregdifferentiation (135,136). Whereas, the involvement of Tregs in the adjuvant effect of the holotoxins still requires further investigation, it is nevertheless already clear that CT exerts a strong adjuvant effect even in IL-10–/– mice, suggesting that Tr1 IL-10-producing cells are not critically involved (137). To get a better understanding of the impact of the holotoxins on the innate and adaptive immune system, global gene-profiling studies were undertaken both in vitro and in vivo. Unfortunately, all of these studies used mixed populations of target cells or even intact tissues to dissect which genes were up- or downregulated following exposure to the holotoxins or mutant derivatives (138–140). The message, though, is clear that many gene families were affected by these molecules. Of more than 100 regulated genes in lymphocytes and

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monocytes, several were encoding immunomodulating molecules such as cytokines, chemokines, chemokine receptors, and co-stimulatory molecules. But, also genes encoding regulators of cell cycle progression, transcription factors, and G-proteins were among the reported genes (138,140,141). The nontoxic LTK63 mutant was found to behave as an airway infection mimic because of the pattern of regulated genes following intrapulmonary administration in mice (138). Several classes of host defense genes were upregulated and T cell–dependent recruitment was triggered in the lung after administration of LTK63, suggesting that the mutant affected host resistance against respiratory infections by modulating both innate and adaptive immune mechanisms. More refined and focused studies, using this approach, may substantially extend our understanding of adjuvant mechanisms in vivo. It would be advantageous, of course, if such studies could identify profiles for the different adjuvants of up- and downregulated genes that correspond to the function in vivo. This way simple screening of gene profiles in target cells or tissues would help in determining immunomodulating potential in newly constructed adjuvants. The fact that holotoxin adjuvants can act to promote the migration of DC and T cells into the tissues after administration was substantiated in the above-mentioned study. LTK63 given to the lung stimulated migration of T cells into lung tissue. Moreover, feeding mice with CT induced a rapid and transient mobilization of a new CD11cþCD8 DC subset near the intestinal epithelium (142). This recruitment was associated with an increased production of the chemokine CCL20 in the small intestine and was followed by a massive accumulation of CD8int/– DCs in MLN, which were also found to stimulate naive T cells more potently than DCs from control mice (40). Previously CT, but not CTB, was shown to upregulate the CXCR4 and CCR7 chemokine receptors, which can have fundamental importance for the adjuvant function, as these receptors favor the migration of immunocytes to the lymph nodes and spleen and promotes the attraction between DC and naive T cells (143,144). Milling et al. recently reported that oral immunization with LT adjuvant in rats augmented CCR7-expression on gut DC migrating to the mesenteric lymph nodes and these cells expressed CD25 and were more effective at stimulating T cells (40). The holotoxins act to upregulate the expression of costimulatory molecules on APCs. In DCs, CD80, CD86, and OX40 were found to be potently upregulated in vitro, while CD40, CCR5, and ICAM-1 were less expressed (130,145,146). To some extent this has also been confirmed in macrophages and B cells exposed to CT (146,147). However, some of the effects of CT were reported after stimulation also with LPS in vitro, while only a few studies have documented effects on CD80, CD86, and CD83 on APC exposed to CT alone (144,147). Whereas in mice no effect on MHC class I or II expression has been documented, the effect in human DCs has shown upregulated expression of HLA-DR molecules and CD80 (143). As mentioned above, the upregulation of IL-10 in exposed DC suppresses the induction of Th1-cells and promotes the development of Treg cells (145). Yet, another pathway for suppression of T-effector cell functions was described by Vendetti et al., as these authors demonstrated increased expression of CTLA4 and reduced expression of CD28 in CT-exposed human T cells (148). Thus, suppression could come from increased development, and function of Treg cells as the relative expression of CTLA4 and CD28 in activated naive T cells

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would impact on the differentiation of these subsets. However, to reconcile the effects on Tregs with an adjuvant function of the holotoxins in vivo awaits further research and analysis. Recombinant B-subunits have been reported to cause the selective depletion of CD8þ T cells from cultures of lymph node cells (149) by induction of apoptosis, lead to the upregulated expression of activation markers on B cells (150,151), and modulated cytokine production and antigen presentation by monocytes/macrophages (152,153). Truitt et al. (154) compared the effects of LT-B on naive, mitogen-activated, and alloantigen-specific CD4þ and CD8þ T cells in vitro. They reported that LT-B preferentially inhibited T-cell responses to TCR crosslinking with monoclonal antibody and to allogeneic histocompatibility antigens in a dose-dependent manner. GM1 binding was essential for this effect. The primary mechanism of T-cell suppression appeared to be rapid induction of apoptosis, with CD8þ T-cells being acutely sensitive to LT-B-induced apoptosis (compared with CD4þ T cells) and naive cells more sensitive than activated cells. By contrast, Yamamoto et al. (130) examined the direct effects of CT on APCs and CD4þ T cells from mucosal inductive tissues (e.g., Peyer’s patches). In those studies, CT directly affected Peyer’s patch CD4þ T cells activated via the TCR-CD3 complex and inhibited proliferative responses. Further, CT induced apoptosis in CD4þ Th1 cells, an effect not seen with CT-B or a CT mutant lacking ADPribosyltransferase activity (130). Also CT has been found to deplete the CD8þ intraepithelial lymphocyte (IEL) population, which could potentially be part of the adjuvant effect as it may reduce the ability to exert downregulation of local IgA responses if CD8þ T cells are lost due to CT (155,156). However, data at variance with such a mechanism were reported by Grdic et al., who demonstrated that mucosal tolerance induced by fed antigen was kept in wild-type mice but was absent in CD8–/– mice when oral immunizations with antigen and CT-adjuvant were performed, arguing that CT acted as an oral adjuvant even in the absence of CD8 T cells (157). Other studies have suggested that ECs may also play a role in mucosal adjuvanticity of the holotoxins. It is clear that the ECs that line the mucosa are more than an inert mechanical barrier; instead, they play a dynamic role as part of a communications network involving immune and inflammatory cells of the host. Human ECs have been found to express and secrete high levels of pro-inflammatory cytokines (158). One possibility is that these adjuvant/toxin molecules interact with ECs, which then express one or more immunomodulatory factors that are important either in recruiting APCs and immune effector cells or activating those cells, or both. LT induces expression of IL-6, IL-10, IL-1R antagonist as well as IL-1a and IL-1b and low levels of IL-8 by human intestinal epithelial T84 cells. Such induction was totally dependent on the intrinsic ADP-ribosylating activity of the toxin Asubunit as neither an enzymatically inactive mutant, LT (E112K), nor LT-B was able to induce cytokine secretion (159). Oral administration of CT has been shown to increase the number of DCs in the FAE and redirect DC from the subepithelial dome of the Peyer’s patch to the adjacent interfollicular T-cell zone (160). DCs in the subepithelial dome express the chemokine receptor CCR6 whose ligand, CCL20 (MIP-3a), is expressed by the FAE. Noteworthy in this context is that DC express tightjunction proteins and are able to open the tight junctions between ECs, send dendrites outside the epithelium, and directly sample the contents of the lumen of the bowel (161).

Hypothetically, adjuvanticity of the holotoxins and their mutants may prove to be useful in exploring the ability of DC to sample antigens and adjuvants in the lumen of the gastrointestinal tract following oral administration (40). It has been shown that CT can increase permeability of the murine intestinal epithelium to low molecular weight peptides (118,162) but not to larger proteins (126). This may be important in explaining the oral adjuvant effect of CT as such an effect would allow antigen to get access to local and systemic innate cells, such as the DCs in the gut lamina proporia (38). Whether this is important, however, has been incompletely studied.

COMBINED ADJUVANT VECTORS ENHANCE IMMUNITY FURTHER To achieve a stronger effect, or a more potent skewing of immune responses, adjuvants may be combined. Thereby, side effects may be limited and the amount of adjuvant and antigen in the vaccine could also be dramatically reduced. Moreover, targeting of adjuvants may allow for enhanced elicitation of specific signaling through, for example, PRRs, because of a higher local adjuvant concentration or better focused administration of the adjuvant. This strategy could even further reduce toxicity and improve safety, while retaining or augmenting strong cell-mediated and humoral immunity. For example, the relative ineffective uptake of CpG-ODN by APC has been found to be helped by a formulation that focuses the CpG to a particle or a carrier protein. Thus, the PLG-CpG ODN combination was found to be an effective adjuvant (163). Another example is the CTB-CpG conjugate, which is an original and innovative combination that renders the CpG especially potent as a mucosal adjuvant. Interestingly this also provides a means to reduce cross-species variability in CpG ODN function and strongly enhanced the TLR9/MyD88-dependent Th1-dominated response in mice (164). A third example is admixing detoxified LTR72 with CpG adjuvant, which gave stronger Th1 responses than seen with CpG or LTR72 alone when given intranasally (165). Whereas the CTA1-DD adjuvant exerts potent mucosal adjuvant effects when given intranasally, it largely fails to enhance immune responses following oral administration. To protect the adjuvant from degradation in the gut we combined it with another mucosal adjuvant, namely immune stimulating complexes (ISCOMS). These particles contain the saponin mixture QuilA as an integrated adjuvant together with phospholipids and cholesterol in a cage-like structure of roughly 40 nm in size (166). Incorporation of CTA1-DD into the ISCOMS was found to greatly enhance the oral adjuvant effect as demonstrated by the strongly enhanced antibody and cell-mediated immune responses (167). ISCOMS are known to primarily act on DC and macrophages, but when combined with CTA1-DD, the complex could be taken up also by B cells, which now effectively could prime naive CD4þ T cells (168). A novel adjuvant with new properties was, thus, developed on the basis of the combination of CTA1-DD and ISCOMS. The combination was highly effective at inducing a wide range of T cell– dependent immune responses, which were only seen with an enzymatically active CTA1-DD molecule, but were lost when a mutant inactive CTA1-R7K-DD molecule was incorporated (168). These examples show that combination adjuvants have many merits and could be developed to reduce significantly the amount of both antigen and adjuvant required in future mucosal vaccines.

Chapter 27: Use of Detoxified CT and LT as Mucosal Adjuvants

CONCLUSION The detoxified CT and LT holotoxins belong to the most promising experimental adjuvants we know of today. However, because of side effects, such as Bell’s palsy, relating to accumulation of toxin in the central nervous system after intranasal use, safety issues have been raised and further clinical exploration of these adjuvants has been somewhat hampered. The CTA1-DD adjuvant, though, without GM1-ganglioside-binding properties, has advanced to be tested in a clinical trial. Studies in nonhuman primates have not indicated any side effects and no accumulation of CTA1-DD in the brain of intranasally treated individuals has been found. Alternative routes of administration still are open for the holotoxins and their detoxified mutant adjuvants. The recent skin-patch technology has boosted an interest in transcutaneous vaccination, and several promising trials are underway. Perhaps the most interesting development, though, is the sublingual route of vaccine administration (169,170). This has provided convincing data on strong cell-mediated as well as humoral immunity adjuvanted by holotoxin. The risk of toxin accumulation in the central nervous system appears to be minimal and, thus, this route should fulfill the safety requirements also for the use of holotoxins as adjuvants. Clinical trials may soon be underway using the detoxified holotoxins as mucosal vaccine adjuvants.

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28 Recent Developments in Nonliving Antigen Delivery Systems Derek T. O’Hagan Novartis Vaccines and Diagnostics, Inc., Cambridge, Massachusetts, U.S.A.

THE SAFETY OF VACCINE ADJUVANTS While complete safety for any medical intervention is impossible, with almost 100 years of accumulated data involving many billions of doses, it is clear that vaccines, including adjuvants, have an excellent safety profile. Recently the safety of longestablished vaccine adjuvants based on insoluble aluminum salts (alum) was readdressed (1). The conclusion was that alum was safe and effective, although local reactions can be common. However, since vaccines are administered to hundreds of millions of individuals on an annual basis, the safety hurdles applied to new vaccines and new vaccine adjuvant technologies will be high, with rigorous evaluation the established practice. It is in this very conservative context that the merits and advantages of novel adjuvants need to be considered. This chapter will focus on a subset of vaccine adjuvants, which can be more accurately described as ‘‘antigen delivery systems,’’ including emulsions, polymeric particles, immunestimulating complexes (ISCOMs), liposomes, and virus-like particles (VLPs). These well-established vaccine adjuvants function primarily through a mechanism that promotes the uptake and processing of associated antigens by important antigen-presenting cells (APCs). However, vaccine adjuvants are generally described by what they do, which is to enhance immune responses to coadministered antigens, rather than what they actually are. Hence adjuvants are a very broad class of compounds, which defy easy descriptions and explanations, so it will be necessary to diversify within this chapter, to include discussions of additional kinds of adjuvants, to allow a full description of the ‘‘delivery system’’–based adjuvants. The mechanisms of action of most adjuvants are often poorly understood, mainly because they are rarely simple, involving many factors, cells, receptors, and processes, which often cooperate in complex cascades with unclear triggers, activators, and feedback loops. Nevertheless, wherever possible within this chapter, simple definitions and descriptions will be offered to add clarity, but this will inevitably result in some necessary over simplifications. Any review on the future development of nonliving antigen delivery systems, inevitably has to begin with a discussion of safety. Many hundreds of different adjuvants have been described in preclinical studies over many years, but only a very few have progressed as far as inclusion into licensed vaccine products, which have survived in the market place. The principal reason for the slow progress of adjuvant development is safety. Approximately 70 years passed between the licensure of insoluble aluminum salts as an adjuvant for vaccines in the 1920s until the licensure of MF59 in the 1990s.

In addition to safety concerns, obstacles in manufacturing have contributed to the slow development of new vaccine adjuvants, including scarcity and high cost of certain raw materials, problems in scale-up, and lack of process reproducibility. However, while it has been possible to overcome many of the manufacturing problems, the issue of adjuvant safety has proven more intractable and often impossible to resolve, despite the expenditure of enormous resources.

THE NEED FOR NEW AND IMPROVED VACCINES There is a clear need for the development of new vaccines against a number of infectious diseases for which vaccines are not yet available, or are inadequate, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), group B Neisseria meningitidis, tuberculosis (TB), and malaria. Unfortunately, these pathogens have proven exceptionally difficult to control, and novel approaches are required. While new technologies are playing a role in the development of vaccines against these difficult targets, including genome-based antigen discovery (2), new vaccine adjuvants also have a key role to play, through their ability to enhance and diversify immune responses (Table 1). New vaccines may also be needed to protect against a number of emerging or reemerging infectious diseases, including Ebola, Hanta, and Dengue viruses. In addition, improved vaccines are necessary to protect against the emergence of pandemic strains of influenza and the continued growth of drug resistant organisms. Moreover, vaccines may be required to protect against the threat of bioterrorism (3). Moving beyond the traditional use of vaccines to prevent infectious diseases, there is an increasing awareness that infectious agents are often the cause of chronic diseases, which might be prevented or treated with novel vaccines. The ability of adjuvants to activate and manipulate the immune response will almost certainly be key to the successful development of therapeutic vaccines against infectious organisms and other causes.

ALTERNATIVE APPROACHES TO IMPROVED VACCINE ADJUVANTS Vaccine adjuvants were first described by Ramon more than 80 years ago (4) and have been used to improve the immunogenicity of most nonliving vaccines ever since. Although the role of an adjuvant is to improve the immunogenicity of antigens, and indeed, this is how they are defined, adjuvants are often included in vaccines to achieve a range of much more specific

Chapter 28: Recent Developments in Nonliving Antigen Delivery Systems Table 1 The Role of Adjuvants in Vaccine Development 1. 2. 3. 4. 5. 6. 7.

Increase antibody titers, e.g., bactericidal, opsonizing or neutralizing antibodies. Decrease the dose of antigen. Decrease the total number of doses. Overcome competition in combination vaccines. Enhance responses in the young or old. Increase the speed and duration of the response. Induce potent cell mediated immunity, including Th1 responses.

effects (Table 1). Historically, adjuvants have been crucial to the development of vaccines, and they are likely to prove even more so in the future. Many vaccines currently in development are comprised of highly purified recombinant proteins, or peptides, usually representing subunits of pathogens. Unfortunately, these vaccines lack most of the features of the original pathogen from which they were derived and are often poorly immunogenic. Therefore, the need for vaccine adjuvants is great. The preferred strategy for the development of new generation vaccines is to add highly purified synthetic adjuvants, which will activate only the elements of the immune response required for protective immunity against the pathogen and will not trigger a more generalized activation of the immune response. Generally, vaccines comprised of attenuated live organisms or whole inactivated organisms do not require adjuvants, as these are sufficiently similar to the native pathogens and usually comprise many inherent adjuvant active molecules. Since adjuvants are defined by the effects they achieve rather than what they actually are, a diverse range of compounds and materials can achieve an adjuvant effect. To define more precisely how adjuvants achieve their effects, it is necessary to reduce the complexity of the immune response down to some simple basic concepts. One concept is to consider the ‘‘signals’’ necessary to induce a successful immune response. With this approach, it is possible to define how adjuvants make important contributions to vaccines, and one can place different kinds of adjuvants into broad groupings to understand how they achieve their effects. The signals necessary for a successful immune response to a vaccine can be summarized as follows: Signal Signal Signal Signal

1—antigen 2—costimulation of immune cells, including APC 3—immune modulation/manipulation 0—activation of the innate immune response

Adjuvants contribute directly to all these signals, but different adjuvants do so in different ways. Some adjuvants are better defined as antigen delivery systems; these are particulate carriers to which antigens can be bound, adsorbed, or associated. This allows the antigens to be stabilized against degradation and clearance, and allows them to be present for extended periods of time. The long-established adjuvant, alum, which is based in insoluble aluminum salts, is thought to work predominantly in this way. Although speculation continues more than 70 years after approval, it is still not entirely clear how alum works (5–7). Nevertheless, it is thought that antigen delivery system–based adjuvants often prolong signal 1 by making the antigen present for extended periods. Prolongation of signal 1 has also been called a ‘‘depot effect,’’ in relation to the mechanism of action of alum. It has been shown that the duration of antigen persistence is important in triggering

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protective T-cell responses (8). Because antigen delivery systems are generally particulates with similar dimensions to pathogens, they are usually taken up efficiently by phagocytosis into APC, the key cells involved in immune response induction. Hence, delivery systems can also contribute to signal 2, through indirect activation of APC due to the particulate uptake process and can sometimes more directly activate APC. In contrast to delivery systems, immune potentiators are a different broad class of adjuvants, which generally exert direct stimulatory effects on immune cells (signals 2 and 3) and can also initiate the immune response through direct activation of innate immunity (signal 0). Although immune potentiators are a very broad class of materials, typical immune potentiators are purified components of bacterial cells or viruses, or synthetic molecules, which mimic their structure. Consequently, they are recognized as ‘‘danger signals’’ by the cells of innate immunity and can be said to express ‘‘pathogen-associated molecular patterns,’’ or PAMPs. There are a number of receptor systems present on and in innate immune cells, which are present to ‘‘sense’’ when an organism is infected. Once these receptors are activated by their ligands, the cells respond accordingly through activation of the innate immune response, which provides a first line of defense against pathogens. These cell-associated receptors, which have been termed pattern recognition receptors (PRRs), have specificity for PAMPs and act to initiate innate immunity. It has recently been proposed that a ‘‘trinity’’ of pathogen sensors is present to activate the innate immune response following exposure to pathogens; namely, the NOD-like receptors, the RIG-like receptors, and the Toll-like receptors (TLRs) (9). Although diverse in their expression patterns and response to different bacterial and viral components, it is apparent that there is significant complementation and overlap between these PRRs in terms of the consequences of individual receptor activation (10). Furthermore, it is increasingly apparent that these and perhaps additional not yet recognized PRR work in harmony to ensure the appropriate activation of the adaptive immune response and the necessary level of immunological activation and memory. The key cells responsible for these activities are the various populations of professional APC, the dendritic cells (DCs), which have a key role to play in linking innate and adaptive immune responses. Peripheral immature DCs, in particular, possess highly effective mechanisms to detect, capture, and respond to pathogens, which may have breached the protective barriers of the skin and mucosal surfaces. Such an encounter triggers maturation and migration of the DCs to key areas of the local lymph nodes, where they can interact with T cells to initiate adaptive immunity. However, DCs are a very heterogeneous population of cells, with different subtypes responsible for different roles in the immune response at different stages of their life cycles (11). The most well known of the PRRs associated with DCs are the rTLR family (12), which recognize diverse components derived from bacteria and viruses and play a key role in initial proinflammatory responses following pathogen exposure. So far, 13 TLRs have been identified in mammals (13), which recognize different microbial components. While some TLRs are located within cell membranes (TLR1, 2, 4, 5, and 6), others are situated within the endoplastic reticulum and endosomes (TLR3, 7, 8, and 9) and recognize various forms of nucleic acids. The expression of TLR can be distinctive to particular cell types of the immune system, or even to nonimmune cells, and TLR expression may be altered in response to cytokines or the presence of distinct kinds of pathogens. In addition to the

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Table 2 A Classification System for Vaccine Adjuvants Antigen delivery systems (particulate carriers)

Immune potentiators (activators of innate immunity)

Combination adjuvant approaches (delivery system þ immune potentiator)

Alum Emulsions Liposomes Microparticles Nanoparticles Virus-like particles (VLPs) Virosomes Calcium phosphate Tyrosine Polyphosphazenes

Natural MPL dsRNA DNA oligonucleotides (CpG, ISS, etc.) Fimbriae MDP and derivatives Synthetic MPL derivatives Quils (e.g., QS21) Small molecule immune potentiators (SMIPs)

MF59 (þ MTP-PE, CpG, etc.) IC31 Alum þ MPL (AS04) ISCOMs Emulsion þ MPL þ QS21 (AS02)

Abbreviations: ISCOMS, immune-stimulating complexes; MDP, muramyl dipeptide; MPL, monophosphoryl lipid.

TLR, a network of cytosolic PRR has also been identified, including the nucleotide-binding oligomerization domain (NOD) receptors (14), which recognize peptidoglycan. Moreover, elegant and sophisticated models are beginning to emerge to explain how the various PRRs might coordinate immune activation signals (10), but these are beyond the scope of this chapter. Nevertheless, it should be clear that a more full understanding of the PRRs, their natural ligands, their dynamics of interactions, where and how they occur, what are the consequences, and so on, will contribute greatly to the design and practical utility of more potent, specific, and safer adjuvants. Many well-established vaccine adjuvants, including monophosphoryl lipid A (MPL), double-stranded RNA (dsRNA), lipopolysaccharide (LPS), lipoproteins (LPPs), and bacterial DNA (CpG sequences) are effective because they are recognized by PRR on innate immune cells. Hence, many adjuvants activate immune responses because they are PAMPs, and these kinds of adjuvants can be classified as ‘‘immune potentiators.’’ This allows a broad and simple classification of adjuvants into delivery systems and immune potentiators (Table 2). If this simplistic classification of delivery systems and immune potentiators is linked to the geographical concept of immune reactivity in which antigens that do not reach local lymph nodes do not induce immune responses (15), it allows a simple definition of the role and mechanism of action of many adjuvants. The role of a delivery system is to enhance the amount of antigen reaching the cells responsible for the immune response, while immune potentiators activate these cells and steer the immune response. Nevertheless, these simple definitions tend to breakdown when immune potentiators are included into delivery systems to focus their effects onto the APC, to maximize their potency, and to minimize their effects on nonimmune cells (Table 2). Hence, delivery systems can improve the therapeutic ratio of immune potentiators, reduce the dose needed, and improve their specificity and safety. New generation vaccines will increasingly comprise recombinant antigens, immune potentiators, and delivery systems working in harmony and exploiting synergies (Fig. 1).

Figure 1 Optimal new generation vaccines.

vaccines in North America (16,17). Alum can be a successful adjuvant, particularly if protective immunity is mediated by antibodies, and if the antigen is already a potent immunogen, for example, for tetanus and diphtheria toxoids. However, in comparative studies with many new generation adjuvants, alum is generally a weak adjuvant, particularly when used with recombinant antigens. Although alum salts have been used for many years as adjuvants, it is still not entirely clear how they work. Alum certainly promotes signal 1, due to adsorption of antigens onto the insoluble aggregates of aluminum, but the slowly dissolving salts also provide some degree of local inflammation. In mice, the adjuvant activity of alum was shown to be mediated by a direct effect on cells expressing Gr-1, which may be granulocytes or a monocyte subset (18). While alum clearly has an important role to play in many existing vaccines, and may still prove sufficiently potent to allow the development of some new generation vaccines, there is a general awareness that more potent adjuvants will be required to allow the development of most new generation vaccines, particularly against the most difficult targets.

The Emulsion Adjuvant MF59 PARTICULATE ANTIGEN DELIVERY SYSTEMS Aluminum Salts Although many adjuvants have been extensively evaluated in preclinical and clinical studies, only insoluble aluminum salts (generically called alum) have so far been included in licensed

In the 1980s, a number of groups were working on the development of novel adjuvant formulations, including emulsions, ISCOMs, liposomes, and microparticles (19). These approaches had the potential to be more potent and effective than the established alum adjuvants. Often the novel adjuvant formulations contained immune potentiators of natural or synthetic

Chapter 28: Recent Developments in Nonliving Antigen Delivery Systems

origin. However, the inclusion of immune potentiators often raised concerns about the safety of the formulation and indeed, has generally restricted the further development of many of these novel approaches. Nevertheless, based on the long history of the use of emulsions as adjuvants, including the widely used Freund’s incomplete adjuvant (FIA), several groups investigated the development of emulsion formulations as adjuvants for use in humans. Scientists at Syntex developed an oil-in-water (o/w) emulsion adjuvant using the biodegradable oil, squalane, and used this as a delivery system for a synthetic immune potentiator called N-acetylmuramyl- L -threonyl- D -isoglutamine (threonyl-MDP) (20). This o/w emulsion adjuvant was called the Syntex adjuvant formulation (SAF). A closely related immune potentiator to threonyl-MDP, N-acetyl-L-alanyl-Disoglutamine (MDP), had been originally identified as the minimal structure isolated from the peptidoglycan of mycobacterial cell walls, which had adjuvant activity (21). However, the parent MDP compound was pyrogenic and induced uveitis in rabbits (22), making it unacceptable as an adjuvant for human vaccines. Therefore, various derivatives of MDP were synthesized in an effort to identify a molecule with an acceptable toxicology profile, which retained adjuvant activity, including threonyl-MDP. MDP and its related compounds were later shown to activate immune cells through interaction with NOD, which acts as an intracellular recognition system for bacteria, and is a PRR (14). In addition to threonyl-MDP, SAF also contained a pluronic polymer surfactant (L121), which was included to help bind antigens to the surface of the emulsion droplets. However, clinical evaluations of SAF as an adjuvant for an HIV vaccine showed it to have an unacceptable profile of reactogenicity (23). As an alternative adjuvant formulation to SAF, Chiron scientists initially developed an o/w emulsion based on the biodegradable oil, squalene, as a delivery system for an alternative synthetic MDP derivative, muramyl tripeptide phosphatidylethanolamine (MTP-PE). MTP-PE had a phospholipid tail attached to it, to allow it to be more easily incorporated into lipid-based formulations, particularly liposomes, and to reduce toxicity (24). Unfortunately, clinical testing showed that emulsions of MTP-PE also showed an unacceptable degree of reactogenicity, making them unsuitable for routine clinical use (25,26). Nevertheless, these studies highlighted that the squalene o/w emulsion alone, without the added MTP-PE immune potentiator, was well tolerated and induced comparable immune responses to the emulsion containing the immune potentiator (26,27). Hence, these observations resulted in the development of the squalene-based o/w emulsion alone as an adjuvant, which was called MF59. The composition of MF59 is shown in Figure 2. The small droplet size of MF59 emulsion, generated through the use of a high-pressure homogenizer, called a microfluidizer, in the preparation process, was crucial to potency, but also enhanced stability and allowed the formulation to be sterile filtered. MF59 emulsion adjuvant, without additional immune potentiators, proved sufficiently potent and safe to allow the successful development of a new generation influenza vaccine containing this adjuvant (28). Hence the experience with MF59 showed that o/w emulsions alone, without additional immune potentiators could be highly effective adjuvants with an acceptable safety profile. Moreover, the early clinical experience with MF59 also served to highlight the need for careful selection of immune potentiators, should it prove necessary to include them in adjuvant formulations.

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Figure 2 The composition of MF59 adjuvant.

The Mechanism of Action of MF59 Adjuvant Early studies designed to determine the mechanism of action of MF59 focused on the possibility of establishing a depot effect for coadministered antigens, since there had been suggestions that emulsions may retain antigen at the injection site. However, it was shown that an antigen depot was not established at the injection site with MF59, and that the emulsion was cleared rapidly (29). The lack of an antigen depot was confirmed in later studies (30), which also established that MF59 and antigen were cleared rapidly. Subsequently, it was thought that perhaps the emulsion acted as a ‘‘direct delivery system’’ and was responsible for promoting the uptake of antigen into APC. This was linked to earlier observations with SAF emulsion, which contained a pluronic surfactant and that was thought to be capable of binding antigen to the emulsion droplets (20). However, studies with recombinant antigens showed that MF59 was an effective adjuvant, despite no evidence of association of the antigens to the oil droplets. Moreover, an adjuvant effect was still observed if MF59 was injected up to 24 hours before the antigen, or up to 1 hour after, confirming that direct association with the emulsion was not required (29). Nevertheless, administration of MF59, 24 hours after the antigen, resulted in a much reduced adjuvant effect, suggesting that the emulsion was activating immune cells, which were able to better process and present the coadministered antigen. A direct effect on cytokine levels in vivo following administration of MF59 has also been observed, supporting the theory of immune activation (31). Moreover, more recent studies have confirmed the ability of MF59 to have a direct effect on immune cells, triggering the release of chemokines and other factors responsible for recruitment and maturation of immune cells. Hence, although the exact mechanism of action of MF59 adjuvant remains to be better defined, it appears to function

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predominantly as a delivery system, to promote the uptake of coadministered vaccine antigens into APC (32,33). However, there does not appear to be a need for the antigen to be directly associated with the emulsion droplets. Rather, MF59 recruits and activates APC to the injection site, which take up and process coadministered antigens. It has been found that MF59 activates human monocytes and granulocytes in vitro. On monocytes, MF59 leads to increased endocytosis, enhanced surface expression of MHC (major histocompatibility complex) class II and CD86, and downregulation of the monocyte lineage marker CD14. These are phenotypic changes consistent with differentiation toward DC lineages. MF59 also induces monocytes and granulocytes to produce chemokines, including CCL2 (MCP-1), CCL4 (MIP-1b), and CXCL8 (IL-8), which are all involved in recruitment of immune cells from the blood into peripheral tissue. In experimental conditions where monocytes differentiate into DC by addition of GM-CSF and IL-4, the presence of MF59 enhances the acquisition of a mature DC phenotype, as monitored by the expression of MHC II, CD86, and CD71. On maturing DC, MF59 leads to an earlier and overall higher expression of the maturation marker CD83 and the chemokine receptor CCR7, which is crucial for homing of DC from peripheral tissue into lymph nodes. Hence, following parenteral vaccination, MF59 increases recruitment of immune cells into the injection site, accelerates and enhances maturation of monocytes, augments Ag uptake, and facilitates migration of differentiating monocytes into the draining lymph nodes. Consequently, MF59 creates a local immune stimulatory environment within the muscle, following immunization, which greatly enhances immune responses to coadministered antigens. The Composition of MF59 MF59 is a low-oil-content o/w emulsion. The oil used for MF59 is squalene, which is a naturally occurring substance found in plants, in the livers of a range of species, including humans, and is a component of the secretions of the sebaceous glands in humans. Moreover, squalene is an intermediate in the human steroid hormone biosynthetic pathway and is a direct synthetic precursor to cholesterol. Therefore, squalene is biodegradable and biocompatible. Eighty percent of shark liver oil is squalene, and shark livers provide the natural source of the squalene, which is used to prepare MF59. MF59 also contains two nonionic surfactants, Tween 80 and Span 85, which have been used in other biomedical products and here, are designed to optimally stabilize the small emulsion droplets. Although single vial formulations can be developed with vaccine antigens dispersed directly in MF59, MF59 can also be added to antigens immediately prior to their administration. Even though a less favorable option, combination prior to administration may be necessary to ensure optimal stability for some more labile antigens. Manufacturing of MF59 Details of the manufacturing process for MF59 have previously been described (34). The process involves dispersing Span 85 in the squalene phase and Tween 80 in the aqueous phase, before high-speed mixing to form a coarse emulsion. The coarse emulsion is then passed repeatedly through a microfluidizer to produce an emulsion of uniform small droplet size (*165 nm), which can then be sterile filtered and filled into vials. Methods have also been published describing the preparation of MF59 at small scale, for use in research studies (35).

Preclinical Experience with MF59 Preclinical experience with MF59 is extensive and has been reviewed on several occasions previously (34,36,37). MF59 has been shown to be a potent adjuvant in a diverse range of species, in combination with a broad range of vaccine antigens, to include recombinant proteins, isolated viral membrane antigens, bacterial toxoids, protein polysaccharide conjugates, peptides, and VLPs. MF59 is particularly effective for inducing high levels of antibodies, including functional titers (neutralizing, bactericidal, and opsonophagocytic titers), and is generally more potent than alum. A preclinical study that directly compared MF59 and alum for several different vaccines confirmed that MF59 was more potent, although alum performed well for bacterial toxoids (38). MF59 has also shown enhanced potency over alum when directly compared by protein polysaccharide conjugate vaccines (39) and by a recombinant viral antigen in nonhuman primates (35). In preclinical studies, MF59 is a more potent adjuvant for influenza vaccines, in comparison with various alternative adjuvants. In addition to immunogenicity studies, extensive preclinical toxicology studies have been undertaken with MF59, in combination with a range of different antigens in a number of species. MF59 has shown no evidence of either mutagenic or teratogenic effects, and does not induce sensitization in an established guinea pig model to assess contact hypersensitivity. The favorable toxicological profile for MF59 allowed extensive clinical testing with a number of different vaccine candidates. Clinical Experience with MF59 Adjuvant The largest clinical experience with MF59 has been obtained with the adjuvanted influenza vaccine (Fluad1), which was initially licensed in Italy in 1997, and is now licensed in more than 20 countries. More than 45 million doses of this product have been used in humans. The adjuvanted influenza vaccine was initially targeted for vaccination of the elderly, since conventional vaccines do not provide optimal protection in this age group (40). For this reason, most of the clinical trials with MF59 adjuvanted influenza vaccines have been performed in elderly subjects, in which a significant adjuvant effect has been consistently observed (41). The increased immunogenicity of MF59 adjuvanted influenza vaccine was shown to be particularly important in subsets of the elderly population, which have a higher risk of developing influenza and its most severe complications, including subjects with a low preimmunization titer and subjects affected by chronic diseases (41,42). Additionally, immunogenicity against heterovariant flu viruses was enhanced by MF59, a feature that is particularly beneficial when the vaccine antigens do not match completely those of the circulating viruses (41,43). Importantly, the addition of MF59 to influenza vaccine did not affect the safety profile of the vaccine, which was very well tolerated (41). MF59 was also evaluated as a potential adjuvant for pandemic influenza vaccines and was shown to induce a highly significant enhancement of antibody titers (44,45). Importantly, MF59 also allowed a significant reduction in the antigen dose, an observation that might be very important to increase the vaccine production capacity when a real pandemic occurs (Fig. 3). As already shown for the interpandemic vaccine (41,43,46), broader cross-neutralization against heterovariant pandemic strains was also an additional benefit of an MF59 adjuvanted vaccine (47). This is an important observation, which might favor the use of MF59 adjuvanted pandemic vaccines for stockpiling purposes.

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Figure 3 Clinical evaluation of H9N2 potential pandemic influenza vaccine with MF59 adjuvant.

The clinical testing of MF59 with other vaccine candidates, including HSV and HBV, has provided additional evidence of the safety, tolerability, and potency of MF59 in adults (48–50). The clinical data on the use of MF59 with a range of vaccine candidates has been reviewed by Podda (36). Clinical data on the use of MF59 as an adjuvant for pediatric vaccines has also been obtained, with cytomegalovirus (CMV) and HIV vaccines. Seronegative toddlers immunized with an MF59 adjuvanted CMV gB vaccine showed antibody titers that were higher than those found in adults naturally infected with CMV. Moreover, the MF59 adjuvanted vaccine was well tolerated in this age group (51). Additionally, an MF59 adjuvanted HIV vaccine was evaluated in newborns, born to HIV positive mothers (52–54). The vaccine was very well tolerated and, despite the presence of maternal antibodies, induced an antibody response in 87% of the immunized infants (53,54). Moreover, the MF59 vaccine was significantly more potent than alum for the induction of cell-mediated immune responses (proliferative T-cell responses) against homologous and heterologous strains of HIV (52). Combination of MF59 with Immunopotentiators Although MF59 is generally a more potent adjuvant than alum (55), it cannot be expected to be suitable for all vaccines. MF59 is particularly effective for enhancing antibody and T-cell proliferative responses (34,55). However, it is not a potent adjuvant for the induction of Th1 cellular immune responses, which may be required to provide protective immunity against some viruses and other intracellular pathogens. Nevertheless, Th1 immunopotentiators, including CpG oligonucleotides (56) have been successfully added to MF59 to improve its potency and to alter the kind of immune response induced (57). Although the formulation of MF59 can be modified to promote

the association of CpG with the oil droplets (57), more recent studies suggest that this may not be necessary, and simple addition of CpG to MF59 may be sufficient. However, careful choice is needed in considering which immune potentiators to add to MF59 emulsion and how best to formulate them. Early experience in the clinic showed that an alternative PAMP immune potentiator, MTP-PE, added to MF59 gave an unacceptable level of reactogenicity (25,26). Although preclinical studies had shown that the potency of MF59 was enhanced by the inclusion of MTP-PE (58) and that this combination appeared to be safe, the available animal models were not able to predict the poor tolerability of MTP-PE in humans. In addition to immune potentiators, alternative delivery systems, including microparticles can also be added to MF59 to enhance its potency (59). However, the level of enhancement achieved would need to be highly significant and probably enabling for vaccine efficacy, to justify the development of such a complex approach. The Use of MF59 in Prime/Boost Settings As an alternative to the inclusion of immune potentiators in MF59 to promote a more potent Th1 response, MF59 can also be used as a booster vaccine with recombinant proteins, once a Th1 response has already been established by immunization with DNA (60). Recently, this strategy has been shown to be promising for the development of a vaccine against HIV, since all arms of the immune response, including cytotoxic T lymphocyte (CTL) responses, T helper responses, and neutralizing antibodies are induced by this combination, prime/boost immunization approach (61–64). A similar approach of DNA prime, followed by protein antigen boost in MF59 has also shown promise in nonhuman primates as a vaccine strategy against HCV (65). Alternatively, protein antigens in MF59 can

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also be used to boost Th1 responses primed by immunization with attenuated viral vectors. The concept of an attenuated viral vector prime followed by MF59 boost has been established in the clinic using canarypox vectors, as a strategy for both HIV (66) and CMV (67). Studies are also showing very encouraging preclinical data with alternative viral vectors, including alphaviruses and adenoviruses. Future Perspectives on the Use of MF59 The encouraging safety and tolerability profile of MF59, in combination with immunogenicity data, suggest that MF59 is an appropriate adjuvant for use in pediatric populations. The stronger adjuvant effect of MF59 in comparison to alum in newborn infants immunized with an HIV vaccine has established the basis for further use of MF59 in this population. Moreover, preclinical data has firmly established that MF59 is a more potent adjuvant than alum for a wide range of vaccines, including recombinant proteins and protein polysaccharide conjugates. Moreover, if necessary, MF59 may be combined with various immune potentiators to enable the development of more complex vaccines, for example, against HCV, and/or HIV, which may also require the use of a prime with DNA or viral vectors.

VIROSOMAL VACCINES An alternative antigen delivery system that is also included in a licensed influenza vaccine in Europe is called immunopotentiating reconstituted influenza virosomes (IRIV), or more simply ‘‘virosomes.’’ Virosomes represent a modification of an established drug delivery approach in which phospholipids are used to prepare vesicles, called liposomes, which have been used as delivery systems for a variety of entrapped drugs. For example, liposomes are used as delivery systems for anticancer drugs in marketed products. Virosomes are prepared by detergent disruption of influenza virus to free the viral membrane glycoproteins, followed by addition of phospholipids to allow vesicle formation, and removal of the detergents. Hence, the membrane antigens from influenza virus, mainly hemagglutinin, are presented in a particulate structure of similar size to the native virus. However, in contrast to the MF59 adjuvanted vaccine, which is focused on the elderly population who need an improved influenza vaccine due to immunosenescence, virosomal vaccines are used in all age groups. Virosomal vaccines appear to represent an alternative approach to inactivated whole virus influenza vaccines, which were originally introduced in the 1960s but have subsequently been largely replaced by subunit vaccines that are more highly purified and better tolerated. Although the virosomal influenza vaccines appear to be better tolerated than the original inactivated flu vaccines, there is limited evidence to suggest that they are actually more immunogenic than conventional influenza vaccines. When virosomal flu vaccines were directly compared with the MF59 adjuvanted vaccine, it was concluded that MF59 induced more potent immune responses (68). In addition, the safety profiles of virosomal and MF59 adjuvanted influenza vaccines appear to be comparable, with both showing only mild and transient local reactions at the injection site. Hence, while it is clear that MF59 offers a significant adjuvant effect for influenza vaccines, particularly for pandemic strains, it is less clear that the virosomal approach actually results in a more potent vaccine. Virosomes appear to offer an alternative means to deliver influenza antigens in a particulate structure that is

well tolerated and can be administered to subjects of wide age range. There is significant interest in using the virosomal approach as a basic delivery system for a wide range of alternative vaccines (69), particularly for recombinant or peptide antigens that are poorly immunogenic when used alone.

POLYMERIC MICROPARTICLES FOR VACCINE DELIVERY The adjuvant effect of synthetic microparticles has been known for many years and has been reviewed previously in detail (70). However, many of the kinds of particles used in early studies were nondegradable and consequently, were not appropriate for development for human use. In addition, since antigens were often chemically conjugated to the particles (71), this added a further level of complexity and made commercial development less likely. As an alternative approach, we have used microparticles prepared from biodegradable polymers with surface adsorbed antigens, since it has been demonstrated that organized arrays of antigens are able to efficiently crosslink B cell receptors and constitute a strong activation signal (72–74). In addition, studies have shown that the duration of antigen persistence is important in triggering protective T-cell responses (8), and antigen persistence is enhanced by microparticles, which offer protection against degradation in vivo. The biodegradable and biocompatible polyesters, the poly (lactide-co-glycolides) (PLG), are the primary candidates for the development of microparticles as vaccine adjuvants, since these have already been used for biomedical purposes in humans for many years (75,76). In addition, PLG polymers have been used for the development of a controlled release drug delivery system for a therapeutic protein (77). Advantages of biodegradable microparticles for vaccine delivery include microparticle uptake ensures delivery of antigen into APCs; the multimeric array of adsorbed antigen epitopes on the surface of microparticles can enhance B cell interaction; microparticles are a flexible platform for co-delivery of immune potentiators and antigens; microparticles focus the effects of immune potentiators on immune cells, which may improve their safety profile; and biodegradable microparticles leave no residue in tissues. It has been shown on many occasions that particles of the appropriate size (*1 mm) are taken up efficiently by APC in vitro (78) and in vivo (79). Moreover, microparticles have been shown to be taken up by APC in vivo, which migrate to the T cell area of local lymph nodes and differentiate into DC (80). The physicochemical properties of microparticles, which control their uptake into macrophages include polymer hydrophobicity, surface charge, and particle size (81). It appears that cationic microparticles may be optimal for uptake into macrophages and DC (82). PLG microparticles were first used for the delivery of entrapped antigens in the early 1990s (83,84). In addition to antibody responses, early studies showed that microparticles were able to induce CTL responses in rodents (85,86). This prompted speculation that microparticles may represent an attractive approach for the development of vaccines against tumors (87). However, the majority of early work focused on the use of microparticles for the controlled release of entrapped antigens, with the objective of making single dose vaccines (88,89). Single dose vaccines would be particularly advantageous in the developing world, where access to health care professionals is difficult to achieve. It was believed that controlled release of antigens from microparticles could be used to

Chapter 28: Recent Developments in Nonliving Antigen Delivery Systems

mimic the usual booster doses of childhood vaccines, allowing effective immunization from a single contact. However, problems arose with this concept due to the degradation of antigens during encapsulation and minimal release of antigen from the microparticles (90). Although a variety of approaches have subsequently attempted to stabilize vaccines entrapped in microparticles, these have generally met with limited success (91–94). Hence, despite significant efforts in basic research over many years, microparticles with entrapped antigens have not yet moved forward into clinical trials as controlled release vaccines. As a result of the problems associated with encapsulating vaccine antigens within microparticles, a novel approach was adopted that adsorbs the antigens onto the surface of microparticles. The novel PLG microparticles with adsorbed antigens induced potent antibody and T-cell responses against recombinant HIV antigens in mice (95) and in nonhuman primates (61). In a more recent study, using a recombinant envelope antigen from HIV, it was shown that adsorbing the antigen onto PLG microparticles allowed retention of the antigenic structure, while microencapsulation of the antigen into the microparticles caused extensive antigenic damage and denaturation (96). It has been found that PLG microparticles induce potent antibody responses to adsorbed recombinant antigens from N. meningitidis group B (Table 3). In addition, PLG microparticles were compared with the more established adjuvant, alum, and established that PLG microparticles represent a viable alternative for a range of traditional and new generation antigens. The complete degradability of the PLG polymer is a clear advantage over alum. Preclinical data would suggest that microparticles may have a role to play as an injectable delivery system for recombinant antigens, but they may prove to be particularly attractive as co-delivery systems for antigens and immune potentiators (Fig. 4). In a recent study, it was established that PLG microparticles can be used to simultaneously deliver both an antigen and an immune potentiator either entrapped within the same microparticle (97) or adsorbed to the microparticle surface (98). Hence, one of the advantages of microparticles for vaccine delivery is that they represent a broad and flexible delivery platform, which can be used for both antigens and immune potentiators. Simultaneous delivery of antigens and immune potentiators into the same APC is an attractive concept, which offers the opportunity to focus the activation effects onto immune cells, while limiting the potential to induce adverse events, through inhibiting the systemic distribution of the immune potentiator (99). Charged PLG microparticles can be used as delivery systems for adsorbed immune potentiators, including CpG (98). Microparticle delivery of adsorbed CpG improved the protective potency of an anthrax vaccine (100). Surprisingly, adsorption to microparticles can make nonactive oligos, active as immune potentiators (101). Table 3 PLG Microparticles Are an Effective Delivery System for an Adsorbed Recombinant Antigen From Neisseria meningitidis Serogroup B (MEN B) Formulation

Serum ELISA titer

Serum bactericidal titer

PLG/Men B Alum/Men B PLG/Men B þ CpG Alum/Men B þ CpG Freund’s adjuvant

227,981 50,211 382,610 56,867 253,844

1024 256 16,384 4096 8192

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Figure 4 A recombinant protein from Neisseria meningitidis serogroup B (Men B) expressed in Escherichia coli was purified and adsorbed to PLG (polylactide co-glycolide) microparticles. Mice were immunized on three occasions intramuscularly with the microparticles on days 0, 21, and 35. The microparticles alone induced moderate titers of serum IgG binding antibodies and bactericidal (meningococcicidal) antibodies. The level of antibodies was enhanced by the addition of monophosphoryl lipid A adjuvant to the microparticles (PLG/Men B þ MPL) but was significantly enhanced even further by entrapping the MPL within the microparticles (PLG/MPL/Men B), to ensure co-delivery of antigen and adjuvant.

The approach of adsorbing antigens onto the surface of microparticles has proven sufficiently flexible to enable the delivery of DNA vaccines. DNA vaccines have a number of potential advantages over alternative approaches; they are highly purified, several antigens can be easily included in the same vaccine; they are inexpensive; and they may be suitable for use in the presence of preexisting maternal immunity. However, it is clear that the immunogenicity of DNA vaccines needs to be significantly improved to allow their successful use in humans (102,103). In a number of studies, cationic PLG microparticles with adsorbed DNA induced significantly enhanced immune responses in comparison to immunization with naked DNA in mice (104) and in nonhuman primates (105). In studies designed to determine the mechanism for the enhanced responses observed, cationic PLG microparticles were able to deliver adsorbed DNA into DC, while naked DNA was not (106,107). Microparticles could be used as delivery systems for entrapped DNA vaccines (108). However, like proteins, DNA is also damaged during encapsulation and release from PLG microparticles (109). In addition, it has not been shown that microencapsulation of DNA actually results in enhanced potency for DNA vaccines in comparison to immunization with naked DNA. In contrast, DNA efficiently adsorbs to cationic PLG microparticles, without structural damage, and results in significantly enhanced immune responses in comparison to naked DNA (104,105,110,111). Cationic PLG microparticles with adsorbed DNA have shown protective efficacy in a rodent colon cancer model (112) and also enhanced the protective efficacy of a DNA vaccine against TB (113). Cationic PLG microparticles with adsorbed DNA are currently being evaluated in a human clinical trial as a new generation HIV vaccine and have shown significant potential in nonhuman primates as an HCV vaccine (65). The potential of microparticles for the development of DNA vaccines was recently reviewed (114). Emulsions with a cationic surface have also been used as delivery systems for DNA vaccines (115). Several alternative biodegradable polymers, including polyanhydrides, polyorthoesters, hyaluronic acid, chitosan, and starch have also been used to prepare microparticles for

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antigen delivery (116), as too have polymers which self-assemble into particulates (poloxamers) (117), or soluble polymers (polyphosphazenes) (118). However, the potency, safety, and tolerability of many of these approaches require further evaluation. Although advantages are often claimed for these approaches over the more established PLG, the advantages are often not clear and are rarely demonstrated in comparative studies.

IMMUNE-STIMULATING COMPLEXES ISCOM adjuvants are a particulate complex containing saponin, cholesterol, and phospholipids. The saponins used for ISCOMs have varied over the years, depending on the manufacturer, but is usually a multicomponent fraction extracted from the bark of the Quillaia saponaria tree. Quillaia saponins have been known for many years to exhibit potent immunomodulatory activity, and have been used in animal vaccines, but the crude preparations are not suitable for human vaccines because of toxicity (119). Therefore, better-defined fractions of Quillaia were developed for human use, including QS21 (120) TM and ISCOPREPTM (121). ISCOPREP saponin is a well-defined, multicomponent fraction, which has been included in ISCOM formulations that have been extensively evaluated in humans and appear to be reasonably well tolerated (122). During the manufacture of ISCOM adjuvants, the cholesterol interacts with saponin to form an extremely strong bond, which is the basis of the unique particulate structure of ISCOMs, and likely contributes to the stability of the adjuvant (123). The interaction with cholesterol also substantially reduces the hemolytic activity of the saponins and is important for safety. Phosphatidylcholine (PC) was traditionally used in the early ISCOM vaccines, usually egg derived. More recently, dipalmytoylphosphatidylcholine (DPPC) has been identified as the optimal phospholipid to be used for ISCOMs manufacturing and stability. A molecular structure for ISCOMs was originally proposed by Kersten et al. (124), who suggested the adjuvant had a ‘‘soccer ball’’ arrangement with the multiple micelles held together by hydrophobic interactions. In this model, the saponin molecules create pores in cholesterol/DPPC vesicles with only the triterpenoid core of the saponin interacting with the lipid bilayer. An important physical property of ISCOMs is the negative surface charge of approximately 20 mV, which enables the adjuvant to form a stable colloidal dispersion. The 40 nm size and particulate structure of the adjuvant is thought to be important for potency, promoting the delivery of antigens to APC. ISCOM adjuvants have been shown to induce potent humoral and cellular immune responses in all species in which they have been evaluated, including nonhuman primates. The immune responses generated in response to immunization with ISCOM adjuvants have recently been reviewed (119). In the 1990s, clinical studies were performed using ISCOMs as adjuvants for influenza vaccines. These studies involved almost 900 participants and showed the induction of both antibody and cellular immune responses (125,126). However, the ISCOM adjuvant was considered too reactogenic to allow further development for human vaccines. Nevertheless, the adjuvant subsequently underwent extensive optimization, to improve tolerability, and the modified adjuvant has now been used in a number of additional clinical studies. NY-ESO-1, an antigen found on a number of cancer types including melanoma and breast, has been formulated with ISCOMs and evaluated in a clinical trial. The vaccine was safe, well tolerated,

and induced both humoral and cellular immune responses (127). Human papilloma virus (HPV) type 16 E6 and E7 proteins were also formulated with ISCOMs and evaluated in two studies. In the second study, the vaccine was found to be immunogenic (humoral and cellular responses), safe, and well tolerated. An HCV core ISCOM vaccine has also been evaluated, and shown to be safe and well tolerated, while inducing both humoral and cell-mediated immune responses. Hence, it appears that a new generation of ISCOM adjuvants are now available, which are capable of inducing potent immune responses in humans, while being better tolerated than the first generation of ISCOMs.

INTERCELL’S ADJUVANT COMPOSITION Intercell has described a vaccine adjuvant formulation that comprises particulate structures made by combining a cationic peptide with an oligonucleotide that does not contain a CpG motif (128). The adjuvant formulation can be added to antigens of interest and is capable of inducing potent immune responses in preclinical models (129). Recently, this adjuvant formulation was progressed into clinical evaluation as an adjuvant for a new generation TB vaccine and appeared to be well tolerated (K. Lingnau, personal communication). The suitability of this new adjuvant formulation for a wide range of vaccines remains to be established, as does its clinical acceptability for further human use.

VIRUS-LIKE PARTICLES The rationale for the use of VLPs as an antigen delivery system came from observations that a glycoprotein antigen was highly immunogenic when presented to the immune system as an ordered array on the surface of virions but not when presented in a soluble or membrane bound form (72). It appears that only highly repetitive antigens are able to efficiently cross-link surface antigen receptors on B cells and to induce potent immune responses (130). VLPs are nonreplicating virus capsids made from recombinant DNA technology that mimics the structure of native viruses. They are generally formed from a single viral structural protein, which self-assembles to form a defined particulate structure, following expression in mammalian cells, yeast, Escherichia coli, or in baculovirus systems. VLPs are noninfectious, as they lack the viral genome and can sometimes be comprised of more than one viral structural protein. However, the most well-known and commonly used VLPs are expressed as a single protein (HPV, HBV core, or calicivirus). VLPs mimic the native structure of the protein comprising their structure and they are potently immunogenic. VLPs are being explored in many different ways as subunit virus vaccine candidates, they can also be used as carriers for heterologous antigens, co-expressed with the VLP protein, or conjugated after preparation. VLPs are a particularly attractive approach if the native virus cannot be easily grown in culture (e.g., HPV). Potentially, VLPs can stimulate both arms of the immune response, including antibodies and T cells, and have significant safety advantages over live-attenuated viral vectors. Antigens can be displayed on VLPs either as genetic fusions (131), by streptavidin-biotin conjugation (132) or by chemical cross-linking (133), with the density of antigen on the surface of the particle apparently playing a critical role in the induction of strong antibody responses (133,134). VLP-based vaccines have been tested in clinical trials to induce immune

Chapter 28: Recent Developments in Nonliving Antigen Delivery Systems

responses against HPV (135), Norwalk virus (136), and HIV (137). In all these studies, the vaccines proved to be safe, well tolerated, and immunogenic. VLPs from HPV were recently approved as a licensed vaccine (138), and this has ensured that the level of interest in this antigen presentation technology will increase significantly in the near future.

WHAT ARE THE CURRENT OPTIONS FOR IMPROVED VACCINE ADJUVANTS? Although alum and MF59 adjuvants, and virosomes are all included in licensed vaccines, each of these has some limitations. In preclinical models using naı¨ve animals, neither alum nor MF59 adjuvants induces potent T-cell immune responses of a Th1 type, which is defined as the ability of antigen primed T cells to produce g-interferon in response to restimulation in vitro. Th1 cells are thought to be particularly important to provide protective immunity against some pathogens, including malaria, HIV, and HCV. Hence the inability of the adjuvants currently included in licensed products to induce potent Th1 responses is thought to be a limiting factor in our ability to develop vaccines against these and other pathogens. Nevertheless, a broad range of immune potentiators are becoming available, which are more able to enhance Th1 responses in preclinical models (99). The first of these, which has been recently included in a licensed vaccine, is called MPL, and is a natural product that is produced by chemically detoxifying bacterial LPS. LPS, which is also known as endotoxin, is very potent at activating the immune system but is too toxic for human use. However, an extensive program in the 1970s identified a reliable and reproducible process for the detoxification of LPS, to allow it to be used as a vaccine adjuvant without significant adverse effects. MPL was first licensed in Europe in early 2005 for use in populations who responded poorly to the existing hepatitis B vaccine, due to renal insufficiency. The product, FendrixTM, contains the traditional adjuvant alum, to which a recombinant antigen is adsorbed, but also contains MPL. The same adjuvant formulation, with MPL adsorbed to alum, is also undergoing late stage clinical evaluation in other vaccines and likely will gain additional approvals within the next few years. Although MPL has been shown to be a safe and effective adjuvant in clinical trials, alternative new generation adjuvants appear to be much more potent for the induction of Th1 responses. In preclinical studies, synthetic oligonucleotides that mimic signature sequences (CpG) present in bacterial DNA appear to be very potent Th1 adjuvants (139). CpG oligonucleotides are currently undergoing early phase clinical evaluation as new generation vaccine adjuvants. In addition to oligo-based adjuvants, synthetic small molecular weight drugs have also been identified, which are able to induce potent Th1 responses (140).

WHAT IS THE BEST LONG-TERM APPROACH FOR ADJUVANT DEVELOPMENT? Although there are many natural products, often extracted from bacteria and viruses, which directly activate immune cells, there is also an increasing interest in the use of synthetic analogs of these agents. Synthetic analogs often have lower manufacturing costs and can be obtained in a highly purified form, which is often in sharp contrast to the heterogeneous natural products. One of the most interesting classes of compounds, which have the potential to be exploited as new

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generation adjuvants are traditional small molecular weight drugs (140). The discovery that traditional drugs can function as vaccine adjuvants required the use of a new terminology, and these drug-like adjuvant active compounds have been called Small Molecular weight Immune Potentiators (SMIPs). The use of SMIPs as adjuvants allows the exploitation of traditional pharmaceutical synthetic approaches, with all the associated advantages, including the ability to manipulate compound structure to control performance, considerable formulation experience with similar compounds, and for some simple and economical synthesis. Hence, there are numerous advantages that can be realized through the use of SMIPs as adjuvants. Given these advantages and the likelihood that many more diverse families of SMIPs will be discovered, it appears likely that a number of SMIPs will become available, to allow better manipulation and control of the immune response. However, it is also clear that new generation delivery systems will be required for SMIP adjuvants to ensure that they are delivered preferentially to key immune cells and that the immune activation signals are not available to a more broad array of cells, due to diffusion of the drugs away from the injection site. Hence, adjuvant formulations will increasingly comprise one or more potent immune potentiators intended to induce the specific kind of immune responses required and formulated into delivery systems designed to maximize potency and minimize potential for adverse events. In this context, the use of microparticles as delivery systems, which were originally developed for the controlled release of small molecular weight drugs, would appear to be a particularly attractive approach. However, extensive research will be necessary to determine the optimal release profiles for different SMIPs and to determine the optimal site for delivery, within a distinctive intracellular compartment, or extracellularly. The preferential site and dynamics of delivery will likely vary extensively, depending on the specific PRR that the SMIP is designed to activate.

CONCLUSIONS AND FUTURE PERSPECTIVE In the past decade, there have been a number of significant advances in technologies designed to identify, express, and deliver vaccine antigens. As a consequence, many of the vaccine candidates currently under evaluation look very different from traditional vaccines. In particular, there has been a shift away from the use of whole pathogens or inactivated subunits, toward the use of recombinant purified proteins. Although this has improved vaccine safety, it has also resulted in the need to develop novel adjuvants and delivery systems to improve the immunogenicity of these antigens. Many future vaccine candidates will likely contain recombinant protein antigens, purified synthetic adjuvants representing welldefined PAMPs, and a delivery system to ensure that both antigen and adjuvant are targeted efficiently to APC (Fig. 2). Formulation of the vaccine into a delivery system will (i) focus the effects of the immune potentiators onto the key cells of the immune system to enhance potency and (ii) limit systemic distribution of the immune potentiators to minimize their potential to induce adverse effects. Novel adjuvant and delivery technologies will be required to enable the successful development of vaccines against diseases that have not yet yielded to traditional approaches. The identification of cell surface markers on different DC subsets, which may allow targeting of particulate delivery systems to specific DC

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populations to induce optimal immune responses, is particularly interesting in this regard (141). The enhanced potency achieved with a targeted system will have to be significantly greater than the currently available nontargeted particulate delivery systems to justify development. Many of the available particulate carriers are already efficiently targeted to APC by passive uptake approaches. Nevertheless, the potential to target to particular DC subsets remains an appealing concept. Heretofore, therapeutic vaccines, that is, the use of vaccines to ameliorate or modify the course of existing chronic infectious or noninfectious diseases has had little success. Therapeutic vaccines have been evaluated for the treatment of cancers, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. While some of these vaccines aim to induce a strong humoral or cell-mediated immune response against a new target antigen, or to break immunological tolerance against a ‘‘self’’ antigen, others are designed to deviate or suppress an existing response. Adjuvants are a key technology for modifying the immune response, and will have an important part to play if therapeutic vaccines are to be successful. The key will be to balance the competing requirements of effective treatment of disease versus the potential induction of immunopathology, as a consequence of pushing the immune system too far in one direction. The high potential in this area needs to be tempered with an appreciation that safety remains crucial and early failures, particularly if focused on safety issues, could set the field back significantly.

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29 Virus-Like Particles as Vaccines and Vaccine Delivery Systems Kathrin U. Jansen SVP Early Phase Programs, Wyeth Vaccine Research, Pearl River, New York, U.S.A.

Margaret E. Conner Department of Molecular Virology and Microbiology and Veterans Affairs Medical Center, Houston, Texas, U.S.A.

Mary K. Estes Department of Molecular Virology and Microbiology, Houston, Texas, U.S.A.

INTRODUCTION Virus-like particles (VLPs) are nonreplicating virus capsids made using recombinant DNA technology. They are generally formed when viral structural protein(s) are synthesized in eukaryotic or prokaryotic expression systems, and the proteins self-assemble to form particles mimicking the structure of native virions. VLPs are not infectious because they lack the viral genome. They can be formed simply by expression of one viral capsid protein (papillomavirus, parvovirus, calicivirus, hepatitis B core protein, or Qb bacteriophage coat protein) or by coexpression of multiple proteins, which form more complex capsid structures (orbivirus, Ebola and Marburg virus, herpesvirus, and rotavirus). VLPs can be produced for both nonenveloped and enveloped RNA and DNA viruses. They are being investigated as vaccines for a number of diseases and as vaccine or DNA delivery vectors to carry protein/peptide antigens or nucleic acid. VLPs are especially useful in situations where native virions cannot be readily isolated or produced (e.g., papillomavirus, Ebola virus, or human norovirus), where traditional vaccine approaches are not possible (1–6). VLPs are advantageous as vaccines because they can induce both arms of the immune system; they display a large repertoire of antigenic sites, and they display discontinuous epitopes that structurally mimic virus particles. Currently, VLP vaccines are licensed for hepatitis B virus (HBV) and human papillomavirus (HPV, see chap. 86 of this book). In other cases, VLP vaccines are in preclinical testing in animal models, and some have progressed to phase I or II testing. An important advantage of VLPs as vaccines is their apparent high safety compared to live or inactivated vaccine approaches. They are safe to both produce and administer because when produced in baculovirus or yeast, they lack mammalian cell proteins or mammalian-derived pathogens (7). In addition, they are a safe vaccine approach for viruses for which a more traditional attenuated or inactivated vaccine approach is not feasible, because of the serious consequences of a reversion to virulence or failure in inactivation (e.g., HIV or HPV). Because VLPs are noninfectious and do not contain nucleic acid, there is no

possibility of reversion to virulence, integration into the host genome, or spread to nonvaccinated individuals. These are important safety advantages, especially when vaccinating children and immunocompromised hosts. Because of their safety record and immunogenicity, VLPs have been increasingly studied to serve as antigen carriers for either peptides or protein antigens targeting infectious diseases, such as influenza or malaria, with the goal to increase the immunogenicity and effectiveness of vaccines for these targets (8–11). More recently, noninfectious disease therapeutics utilizing VLP carrier technologies have been evaluated, and some have entered clinical development (12–15). VLPs can be produced or expressed in many systems, including mammalian cells, baculovirus, yeast, recombinant vaccinia, Salmonella, E. coli, alphavirus replicons, and plants (7). The production system needs to guarantee correct folding of the VLP structure and adequate yields for commercial production. The system of choice varies depending on the particular VLP being produced. Assembly of some VLPs may require authentic post-translational modifications of viral proteins only possible in eukaryotic cells, or some pathogenic gene sequences may be toxic or inadequate for expression in bacteria or yeast. In these cases, modifications of the expression system or changes in codon usage (16) may overcome initial potential disadvantages.

VIRUS-LIKE PARTICLES AS VACCINES VLPs can deliver a vaccine immunogen with an ordered structure and intact conformational epitopes. Disrupted VLPs or mutant proteins that aggregate, but not self-assemble to form VLPs, are much less or nonimmunogenic (4,17,18). While VLPs are typically more immunogenic than subunit vaccines, they often can still benefit from the coadministration of adjuvants (4,19–21). In fact, all licensed VLPs are administered with an adjuvant. VLPs are typically effective inducers of neutralizing antibodies. For most pathogens, vaccine-induced protection against

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Figure 1 VLPs produced in insect and yeast cells used for vaccine studies in humans and animals. (Top left). Transmission electron microscopy image of hepatitis B surface antigen particles negative-stained with phosphotungstic acid. Scale bar is 200 nm. (Bottom left) Cryoelectron microscopy image of human papillomavirus (L1 type 6) VLPs preserved in vitreous ice (no dilution). Scale bar is 200 nm. (Top right) NV VLPs produced in Sf9 cells and negative stained with ammonium molybdate. Scale bar is 100 nm. (Bottom right) Rotavirus virus 2/4/ 6/7-VLPs produced in SF9 cells and stained with ammonium molybdate. Scale bar is 100 nm. Abbreviations: VLPs, virus-like particles; NV, Norwalk virus. Source: (Top and bottom left) Courtesy of Merck & Co., Inc. and the National Resource for Automated Molecular Microscopy, The Scripps Research Institute; (Top and bottom right) Courtesy of M. Estes, Baylor College of Medicine.

disease is dependent upon the presence of neutralizing antibodies at the time of subsequent exposure to the pathogen. However, an advantage of VLPs is that they can also induce T-cell responses, as measured by bulk culture proliferation assays and cytotoxic T lymphocyte assays, and VLPs can be presented by both class I and II major histocompatibility complex (MHC) pathways. VLPs induce both cytotoxic and helper T cells, frequently without the addition of adjuvant, and can confer protection from virus challenge. However, more potent induction of cytotoxic T-cell responses may occur when adjuvants or other moieties that activate antigen-presenting cells are included in the vaccine (18,22–27). While VLPs are strong inducers of immune responses, they may induce different or more limited immune effector responses compared to responses to native virus infection (28). Different VLPs may induce different immune mediators, even if the VLPs are similar in size and structure (e.g., papillomavirus vs. polyomavirus) (18,29). The route of VLP administration and adjuvant can also affect the immune response. Papillomavirus VLPs administered parenterally induce vaginal IgG (30) but not IgA, while orally administered VLPs induce vaginal IgA (31). Taken together, these data suggest that while presentation of structure is an important parameter of VLP-induced immune responses, other factors also affect the immune response induced by VLPs.

The remainder of this chapter will focus on use of VLPs as vaccines for several diseases, including hepatitis B, human papillomavirus, and enteric infections caused by Norwalk virus (NV) and rotavirus (Fig. 1). These systems are being emphasized because these VLPs have been licensed, are in phase I clinical trials, or have undergone extensive preclinical testing.

Hepatitis B Virus Vaccines Infection with HBV leads to an acute infection and recovery in 95% of adults and 5% to 10% of children. However, the remaining infections result in chronic viral infection. Chronically infected individuals are at risk to develop cirrhosis and hepatocellular carcinoma that may occur 30 to 50 years after initial infection. Hepatitis B surface antigen (HBsAg) purified as particles from plasma can be considered as the first VLP vaccines licensed to prevent an infectious disease, hepatitis, and this vaccine is still being used in the developing world. It has been supplanted in other countries by recombinantly produced HBsAg. These vaccines consist of the small envelope (s) protein and the middle pre-S2 envelope (M) protein assembled into 22 nm particles. The vaccines are administered parenterally by intramuscular injection, are highly immunogenic, and induce protective anti-HBs titers (>10 IU/mL) in more than 95% of healthy children and young adults. The

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efficacy of protection against HBV infection has been proven in large clinical studies of exposed populations, such as homosexual men, health care workers, and infants born to HBsAgpositive mothers. Antibody titers may decline to undetectable levels several years after vaccination, but immunity against clinical disease persists for years, suggesting the existence of immunologic memory (32). On the basis of these results, a booster is not recommended in healthy individuals who are not exposed to a high risk of HBV infection. HBV is the first example of a successful cancer vaccine; it has been shown to reduce hepatocellular cancer in a number of population studies (33,34). The vaccine has been used in millions of individuals and has demonstrated an excellent safety and efficacy record illustrating the high potential for VLP vaccines.

Human Papillomavirus Vaccines Infection with HPV constitutes a serious problem in women’s health worldwide. HPV causes cervical, vaginal, and vulvar cancers; precancerous lesions; and genital warts. Worldwide close to 500,000 women will be diagnosed with cervical cancer annually, and half of them will die from the disease (35). About 80% of the disease burden is in the developing world where most women do not have access to screening programs and high-quality gynecological care. HPV is also responsible for anogenital lesions and cancer in men and has been implicated through epidemiological studies to the etiology of certain oropharyngeal cancers (36). While there are close to 40 HPV types infecting humans, only a few types cause the majority of disease. HPV 16 and 18 are responsible for 70% of all cervical and anal cancers in both women and men, while HPV 6 and 11 cause over 90% of all genital warts in both genders. Recently, two vaccines to protect against HPV infection and disease were licensed and both vaccines are VLPs (see also chap. 86 in this book). As with HBV, the coat protein of HPV when expressed recombinantly self-assembles into VLPs of approximately 50 nm that are highly immunogenic and good inducers of virus neutralizing antibodies (30,37,38). The vaccine licensed first, Gardasil1 (Merck & Co., Inc., Whitehouse Station, New Jersey, U.S.), is composed of four VLPs covering HPV types 16, 18, 6, and 11. The VLPs are produced in baker’s yeast and formulated on aluminum hydroxyphosphate sulfate adjuvant. The vaccine was approved for the prevention of cervical cancer and its precursor lesions, vulvar and vaginal cancers and their precursors, and the prevention of genital warts. In phase III clinical trials, the vaccine was 100% effective against HPV 6, 11, 16, and 18 precancerous lesions (39) and 100% effective against genital warts (40). Immunogenicity in both male and female adolescents was also established (41). The second vaccine, Cervarix1 (GlaxoSmithKline Biologicals, Rixensart, Belgium), is composed of only two VLPs covering HPV 16 and 18. The VLPs are produced in insect cells and formulated on aluminum hydroxide and 3-deacylated monophosphoryl lipid A. In phase III clinical evaluation, the vaccine was 90.4% effective against HPV16/18 cervical intraepithelial neoplasia 2þ (CIN2þ) when a prespecified analysis was used (42). Because Cervarix1 does not contain VLPs against the genital wart type HPVs, it has no efficacy against genital warts. The stunningly good efficacy data for both vaccines and their safety profile provide another example for the potential of VLP-based vaccines, and one can only hope that these vaccines get used widely not only in the developed world but also in developing countries, where such vaccines are the only hope to reduce the cervical cancer burden.

VLP VACCINES FOR ENTERIC VIRUSES (NORWALK VIRUS AND ROTAVIRUS VLPS) Candidate VLP vaccines are being pursued for two enteric viral pathogens, rotavirus and NV. Both viruses cause acute, gastrointestinal infections of humans, and both are also important pathogens in many animal species. Challenges for development of effective vaccines for both rotavirus and NV include the need to induce mucosal immune responses to protect from diarrheal disease, and the need to target the vaccine to young children, particularly for rotavirus. We review briefly below the progress toward development of NV and rotavirus VLP vaccines for use in humans.

Norwalk Virus and Norwalk-Like Viruses (Noroviruses) NV is the prototype virus of the Norovirus (NoV) genus, within the genetically diverse, single-stranded RNA virus family Caliciviridae (43). NoVs are the leading viral cause of epidemic food- and water-borne diarrheal and vomiting illness in all age groups. The NoVs are considered ‘‘emerging pathogens’’ because of the rapidly expanding disease burden attributed to infections with these viruses as new tests for diagnosis have become available. The NoVs are also considered class B biodefense pathogens because of their apparent low infectious dose and potential to cause large outbreaks of waterborne, and possibly airborne, disease. In the United States, NoVs cause an estimated 23 million episodes of illness, 50,000 hospitalizations, and 300 deaths each year (44). NoVs can be transmitted by fecally contaminated food and water and by direct person-toperson contact or through droplets from infected persons. Outbreaks are a particular concern in elderly residents of nursing homes, military personnel, and travelers. The use of new diagnostic assays to detect these genetically diverse pathogens has rapidly determined that these viruses cause significantly more infections than previously recognized, infecting all age groups. The clinical manifestations of NoV infections include sudden onset of vomiting and/or diarrhea, which typically last 12 to 24 hours after a 24- to –48-hour incubation period. The increasing disease burden and significance of infections in selected settings, combined with the discovery that the expressed capsid protein of NV folds spontaneously into VLPs, have stimulated vaccine development (45,46). Studies with NV VLPs also serve as an excellent model to dissect and understand effective strategies for mucosal immunization with nonreplicating antigens because of the following useful properties. First, the VLPs are stable at low pH, so they can be administered orally. Second, they can be lyophilized and stored at 48C in water or phosphate-buffered saline (PBS) for at least three years without degradation. Third, the VLPs are easily made and are obtained in sufficient purity for vaccine evaluation and successful crystallization (47). Finally, NV VLPs are immunogenic when tested in inbred and outbred mice and in volunteers following systemic, oral, or intranasal administration, even in the absence of a mucosal adjuvant and at low doses (5,20,48). On the basis of the preclinical immunogenicity, two phase I studies have evaluated oral administration of two doses of varying amounts (100, 250, 500, and 2000 mg) of NV VLPs without adjuvant to healthy, adult volunteers (5,6). The VLPs were safe and immunogenic. Serum IgG responses to the VLPs were dose-dependent, and all vaccinees given 250 mg responded with serum IgG titers. Most of the volunteers responded after the first dose and showed no increase in

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serum IgG titer after the second dose. All vaccinees developed significant rises in IgA antibody secreting cells after vaccination. Ninety percent of vaccinees who received 250 mg developed a rise in serum IgG anti-VLP antibody, but neither the rates of seroconversion nor titers increased at the higher doses. About 30% to 40% of volunteers developed salivary, fecal, or genital fluid IgA antibody. However, the maximal titers of serum antibody induced by VLP immunization were lower than titers seen following infection with NV. Studies are needed to see whether the immunogenicity of NV VLPs can be further enhanced using mucosal adjuvants. One such phase I study evaluating an intranasally delivered, dry powder vaccine formulation containing NV/VLPs, the adjuvant monophosphoryl lipid A, and chitosan to enhance nasal delivery is ongoing (49). NV is a noncutivatable human pathogen, and there are no small animal models available to study pathogenesis or protection from disease. Therefore, efficacy studies will require reestablishing a human challenge model (5,50).

Rotavirus VLP-Based Vaccines Rotaviruses cause nearly 39% of diarrheal disease in infants and young children worldwide; 30% of all children develop a rotavirus infection before nine months of age and 80% within the first three years of life (51). The peak age of disease of children in developing countries is younger than in developed countries and is frequently between three and six months (52). Rotaviral-related diarrheal deaths are rare in developed countries, but in developing countries cause an estimated annual 450,000 to 705,000 deaths (51). In the United States, approximately three million children are ill annually from rotavirus, and an estimated 700,000 children need medical attention; illness is serious enough in 280,000 children to require an emergency department visit or hospitalization, all at an estimated cost of approximately one billion dollars annually (53). For these reasons, the development of a rotavirus vaccine to prevent severe dehydrating diarrhea in young children is a major global health priority. There are many serotypes of rotavirus that infect children, and multiple serotypes of rotavirus circulate concurrently. Rotaviruses have a dual serotype classification system of P and G types, based on two neutralization antigens VP4 and VP7, respectively (54). Rotavirus disease worldwide has been most frequently associated with G1–G4 types, and these types have been targeted in vaccine development. Immune correlates of protection for rotavirus have not been clearly defined in children or animals, but intestinal antibodies of both IgA and IgG subclasses are thought to be of primary importance in protection (55). Effective, traditional human and human-animal rotavirus reassortant rotavirus vaccines have been licensed for use in children in some countries and are in field trials in developing countries where these vaccines are needed most (56–58). The safety of live attenuated rotavirus vaccines was raised when intussusception (telescoping of the intestine) was associated with the first licensed rotavirus reassortant vaccine (RotaShield1, Wyeth Lederle Vaccines, Philadelphia, U.S.), a vaccine that was subsequently removed from the market (59). While subsequent investigation suggests that intussusception was associated with children who received that vaccine at older ages than recommended (54) and it has not been seen with the current licensed vaccines, there are still lingering safety concerns about live attenuated vaccines. On the other hand,

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rotavirus VLP vaccines have been studied extensively and may become an alternative to the existing vaccines. Rotavirus VLPs are made by coinfecting insect cells with baculovirus recombinants that express rotavirus structural proteins (VP2, 4, 6, and 7). These proteins self-assemble into VLPs that are morphologically and antigenically similar to rotavirus (60). They can be coexpressed in different combinations producing either single-layered (VP2 only) or double-layered VLPs (VP2 and 6), mimicking the innermost middle capsid of rotavirus, or triple-layered VLPs without (VP2, 6, and 7) or with the spike protein (VP2,4, 6, and 7) (60,61). Chimeric rotavirus VLPs are readily formed with individual rotavirus proteins from different rotavirus strains or with one protein from two different rotavirus strains on one particle (62). The immunogenicity of rotavirus VLPs in different combinantions has been extensively evaluated in adult rabbits, adult mice, and gnotobiotic piglets. Some forms have been shown to induce passive protection in neonatal mice and calves (63,64), and active protection in mice, rabbits, and pigs (19,65–67). All the preclinical data with rotavirus VLPs support the testing of rotavirus VLPs in humans. Rotavirus VLPs are immunogenic and at least partially protective by all routes tested. Coadministration of adjuvants increases immunogenicity, lowers the protective dose of VLPs, and may enhance the longevity of the protective immune response. Mucosal administration of rotavirus VLPs to humans provides many advantages, including ease of delivery, no need for needles, and cost especially in the developing world where vaccines are most urgently needed, but where health care funding is limited. Intranasal delivery of VLPs is a very effective route of administration to induce rotavirus-specific antibody in the intestine in two of the three animal models in which it has been tested. Comparisons of doses between oral and intranasal routes of immunization indicate that intranasal delivery of VLPs induces higher levels of protection, and requires up to 10-fold lower doses of VLPs than needed to achieve low to moderate protection when VLPs are administered orally. Intranasal administration of VLPs may be superior to oral administration due to limited degradation in the respiratory compared to the intestinal tract, increased retention of and interaction of VLPs with M cells or lymphocytes, or differences in antigen uptake or processing. Further work is needed to determine if this highly promising vaccination route will be safe for use in humans. Studies are underway to understand the differences in immunogenicity and protective efficacy induced by the two mucosal routes and to determine if modifications to the vaccination protocol or VLPs themselves can reduce the variability in protection and enhance the immunogenicity and protective efficacy of orally administered VLPs. Parenteral immunization with VLPs may be the best route for children in developing countries where successful immunization with oral vaccines is known to be challenging. Vaccine studies in human subjects with the most promising candidates (2/6/7- and 2/4/6/7 VLPs) are long overdue and may help to define which animal models are most predictive of responses in children or pregnant women, the target populations for rotavirus VLP vaccines.

VLPs As Vaccine Carriers Hepatitis B Virus VLPs As discussed in the preceding text, recombinant VLP vaccines for viral diseases have been very successful in preventing infection and disease in humans and animals, and mirror the

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efficacy of live or attenuated virus vaccines (for an additional review of the use of VLPs as immunogens, see also Ref. 7). Since VLPs are recombinantly produced and do not contain any infectious genetic material, they enjoy a very good safety profile, which is exemplified by the vaccines against HBV and HPV. VLPs, while having complex structures, are usually easily and reproducibly manufactured. Unlike live or attenuated vaccines, VLPs can be readily characterized using a number of biophysical and analytical assays. Because of the repetitive nature of the protective epitopes displayed by the VLPs and their size, VLPs are generally more immunogenic than protein subunit vaccines. For all these reasons, VLPs have been increasingly considered as antigen carriers for either peptides or protein antigens, targeting infectious diseases like influenza or malaria, with the goal to increase the immunogenicity and effectiveness of these vaccine targets. More recently, noninfectious disease therapeutics utilizing VLP technology have been evaluated, and some have entered clinical development. VLPs have also been used as carriers for DNA to assess their ability to serve as gene-transfer vehicles (68). The following sections will discuss the utility and promise of VLPs as a carrier technology using just a few examples. VLPs as Antigen Carriers for Infectious Disease Targets The use of VLPs as antigen carriers is not new and was first described about 20 years ago. A selection of VLPs could be useful as antigen carriers. While there are a large number of studies involving VLPs in the context of a number of different infectious diseases, the review of all these studies would exceed the purpose of this chapter. However, we wish to discuss two examples, VLPs as carriers for the influenza M2 peptide and VLPs as carriers for malaria antigens. VLPs as carriers for the influenza M2 protein. Influenza is one of the most significant respiratory diseases worldwide, yet currently licensed inactivated influenza vaccines that today are the best way to protect against the disease are hampered by a number of drawbacks. The vaccines are all based on the protective efficacy of hemagglutinin (HA) and, to a much lesser extent, neuraminidase (NA), which are subject to antigenic drift and shift requiring seasonal updates of the vaccines. The vaccines have only moderate to low efficacy, especially in the target populations that are most vulnerable to the disease, namely, the very young and old. In contrast to HA or NA, the influenza M2 protein, a third integral membrane protein of influenza A, is highly conserved but not very immunogenic. Hence, it may not be subjected to the same immune selection pressure as HA or NA and may therefore be a better protective antigen with the promise of inducing lasting protection over a number of influenza seasons. The antigenic portion of M2 is composed of the extracellular portion of M2, an approximately 25 amino acid long peptide. To increase the immunogenicity of the M2 peptide, it needs to be linked to a carrier. Two VLP carriers, hepatitis B core antigen (HBcAg) and human papillomavirus type 16 (HPV16) have been studied (9–11). In the case of HBcAg, the M2 peptide was spliced into the HBcAg gene sequence, and chimeric VLPs were expressed recombinantly in E. coli (11). In the case of the HPV 16 VLPs, yeast-expressed purified HPV16 VLPs were used to chemically conjugate the M2 peptide using a heterobifunctional cross-linker (10). Both M2 carrier VLP systems induced high-titered-antibody responses, and both were able to protect mice against a lethal influenza virus challenge. Cross-protection against different influenza strains was also observed (11). While these studies

look very promising, to our knowledge, none of these two candidate VLP-based vaccines has reached clinical development. Furthermore, the choice of carrier HBcAg or HPV VLPs may not be optimal. Both carrier VLPs are part of licensed vaccines and, in the context of having heterologous antigens attached to them, may interfere with the efficacy of these existing vaccines. VLPs as carriers for malaria antigens. Malaria, which is caused by four species of the protozoan Plasmodium, is estimated to cause 300 to 500 million cases a year globally, with an associated death rate of 1.4 to 2.7 million. The majority of deaths occur in children in Africa. Plasmodium has a complex life cycle, and vaccine development is targeting a number of them. Several studies have demonstrated that the circumsporozoite (CS) protein of P. falciparum harbors protective epitopes in the immunodominant repeat region of CS that can neutralize sporozoite infection and invasion of liver cells. Several phase 1 and 2 clinical trials have tested different vaccines on the basis of CS peptide protein conjugates or subunit protein vaccines incorporating the protective epitopes. The goal was to find the most immunogenic and effective vaccine construct (8). The studies identified the need to assess additional vaccine delivery systems including VLPs as carriers for CS-protective epitopes and to have these epitopes presented in the context of effective B- and T-cell epitopes. One of these vaccine constructs, ICC 1132 (Malarivax, Coralville, Iowa, U.S.), utilizes the HBcAg fused to a protective B-cell epitope of the CS repeat region and two powerful T-cell epitopes identified from human CD4þ T-cell clones obtained from sporozoite immunized individuals. Small clinical studies in human volunteers showed that the Tcell epitopes were effectively processed and presented in the context of HBcAg VLPs. Two adjuvanted formulations of the chimeric HBcAg VLPs, one with aluminum hydroxide and the other with Montanide ISA 720, a metabolizable oil adjuvant containing a mannide monooleate emulsifier, were tested in phase 1 clinical trials (8). The trials demonstrated that all vaccine formulations were well tolerated and immunogenic. A single immunization of the ISA 720 formulated vaccine was able to achieve comparable titers elicited after three doses of the aluminum hydroxide formulation. While these immunogenicity results using chimeric VLPs were encouraging, they were not sufficient to protect against P. falciparum challenges in humans. It is likely that more than one dose of the vaccine would be needed to induce a protective immune response, and additional formulation work may be required to increase the immunogenicity of these constructs. VLPs as Antigen Carriers for Therapeutics As discussed above, VLPs have been very successful in inducing protective immunity against a number of infectious disease targets. Recently, VLPs were evaluated as antigen carriers in vaccines targeting therapeutic diseases like nicotine addiction (14), allergy (12), hypertension (69), and Alzheimer’s disease (15). The finding that VLPs are potent inducers of immune responses to self antigen (70) has allowed the approach of evaluating VLPs both as carriers for self and nonself antigens. A number of such vaccines have now entered the stages of clinical evaluation. Again, we will focus on just two examples, a VLP-based vaccine against Alzheimer’s disease and nicotine addiction. A VLP-based vaccine against Alzheimer’s disease. The prevalence of Alzheimer’s disease is 67/1000 in the general elderly population (71). Amyloid-beta (Ab) plaque accumulation

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in the brain of Alzheimer’s patients is strongly linked to the pathophysiology of the disease. A number of groups are working on both active and passive immunization strategies to combat this terrible disease. Preclinical as well as clinical data suggest a reasonable potential for such approaches. Initial clinical studies using active immunotherapy with an Ab vaccine known as AN1792 demonstrated some positive effects (72–74), even though the studies were halted due to an unacceptable side effect profile (meningoencephalitis) of the vaccine in approximately 6% of immunized patients. It is believed that the side effects were caused by induction of an inflammatory immune response in response to the vaccination strategy used in this study. VLPs are attractive alternatives for an active vaccination approach since they have been shown to induce high-titered antibodies even in the absence of adjuvants. Furthermore, it was shown that VLPs coupled to an autoantigen failed to induce T-cell responses to the autoantigen itself, yet were able to induce T-cell responses to the VLP carrier. Chackerian et al. (15) tested two VLP carriers, HPV 16 VLPs expressed in 293 TT cells and Qb phage VLPs produced in E. coli for a number of Ab peptide constructs. Both VLP carriers in the absence of adjuvants induced high-titered antibody responses in mice against the Ab peptide components that mirrored the responses against the Ab peptides formulated with adjuvants. The Qb VLPs carrier appeared more immunogenic than the HPV 16 VLP carrier. Unlike the Ab peptide/ adjuvant group, which elicited a predominantly Th1-type inflammatory response, the VLP carrier Ab constructs elicited a solid Th2-type IgG1 response. While this study holds promise, follow-up studies in Alzheimer’s models and primates are needed to explore this approach more fully. A VLP-based vaccine for nicotine addiction. Another example for the usefulness of VLPs is their use as antigen carrier in a vaccine targeting nicotine addiction (14). Globally, tobacco-related deaths are estimated by the WHO to be over 10 million annually. Nicotine is the main culprit responsible for inducing and sustaining dependence and addiction to tobacco products. The vaccine concept is based on the binding of nicotine molecules by anti-nicotine antibodies, thereby preventing nicotine from traversing the blood-brain barrier. This vaccine has passed the preclinical testing stage and entered clinical development (phase II) (75). The VLP carrier in the vaccine consists of the coat protein of the Qb bacteriophage that self-assembles into approximately 25 nm icosahedral structures when expressed in E. coli. Nicotine molecules were covalently linked to the VLPs resulting in approximately 585 nicotine molecules coupled to each VLP. Formulations both with and without aluminum adjuvant were tested in preclinical models and in the clinic. Higher anti-nicotine antibody levels were elicited by the aluminum adjuvant formulated vaccines both in preclinical as well as clinical settings. A phase II randomized and placebo-controlled proof-of-concept study tested the vaccine’s ability to induce continuous abstinence from smoking (75). The study found that only subjects who responded well to the vaccine (high responders) showed a statistically significant continuous abstinence from smoking compared to placebo at six months (1 month after completion of the 5-dose vaccine series), or 22/53 (57%) of subjects in the high-responder vaccine group compared to 25/80 (31%) of subjects in the placebo group. After 12 months, 42% in the high-responder vaccine group compared to 21% in the placebo group still remained abstinent. While these studies are promising, a number of challenges remain. Clearly, the vaccine formulation needs

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improvement to increase the proportion of individuals with high and sustainable anti-nicotine antibodies. The vaccine dose and regimen also deserve attention, since the five doses given one month apart may not be optimal in maximizing both the level of anti-nicotine antibodies as well as the duration of the response.

SUMMARY VLPs have demonstrated exquisite protection and safety against diseases like hepatitis B and cervical cancer caused by HPV. The only two successful cancer vaccines to date (HPV and HBV vaccines) are both based on VLP technology. Promising candidates for parenterally and mucosally administered subunit vaccines to prevent diarrheal diseases also have been identified. Rotavirus VLPs have been extensively tested in animal models and await testing in humans. Norwalk VLPs are immunogenic in both animals and humans but protective efficacy needs to be tested in humans using a human challenge model. The immunogenicity and safety of VLP-based vaccines has invigorated research in VLP-based vaccines against other infectious diseases as well as noninfectious disease targets. VLPs are increasingly considered as antigen carriers in these settings, and promising vaccine results have been demonstrated in preclinical as well as clinical studies.

ACKNOWLEDGMENTS The authors are grateful to their students, postdoctoral fellows, and colleagues who have worked with them to demonstrate the effectiveness of VLPs as immunogens and vaccine carriers and to understand their mechanisms of inducing potent and effective immune responses.

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mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast. J Infect Dis 1997; 176(5):1141–1145. Nardelli-Haefliger D, Lurati F, Wirthner D, et al. Immune responses induced by lower airway mucosal immunisation with a human papillomavirus type 16 virus-like particle vaccine. Vaccine 2005; 23(28):3634–3641. Van Damme P, Van Herck K. A review of the long-term protection after hepatitis A and B vaccination. Travel Med Infect Dis 2007; 5(2):79–84. Chang M-H. Decreasing incidence of hepatocellular carcinoma among children following universal hepatitis B immunization. Liver Int 2003; 23(5):309–314. Montesano R. Hepatitis B immunization and hepatocellular carcinoma: The Gambia hepatitis intervention study. J Med Virol 2002; 67(3):444–446. Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55(2):74–108. D’Souza G, Kreimer AR, Viscidi R, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med 2007; 356(19):1993–1995. Zhou J, Sun XY, Stenzel DJ, et al. Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly of HPV virion-like particles. Virology 1991; 185(1): 251–257. Kirnbauer R, Booy F, Cheng N, et al. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Proc Natl Acad Sci U S A 1992; 89(24): 12180–12184. Joura EA, Leodolter S, Hernandez-Avila M, et al. Efficacy of a quadrivalent prophylactic human papillomavirus (types 6, 11, 16, and 18) L1 virus-like-particle vaccine against high-grade vulval and vaginal lesions: a combined analysis of three randomised clinical trials. Lancet 2007; 369(9574):1693–1702. Garland SM, Hernandez-Avila M, Wheeler C, et al. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007; 356(19):1915–1927. Reisinger KS, Block SL, Lazcano-Ponce E, et al. Safety and persistent immunogenicity of a quadrivalent human papillomavirus types 6, 11, 16, 18 L1 virus-like particle vaccine in preadolescents and adolescents: a randomized controlled trial. Pediatr Infect Dis J 2007; 26(3):201–209. Paavonen J, Jenkins D, Bosch FX, et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007; 369(9580):2161–2170. Green KY, Chanock RM, Kapikian AZ, et al. The Noroviruses. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:949–979. Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999; 5(5):607–625. Jiang X, Wang M, Graham DY, et al. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein. J Virol 1992; 66(11):6527–6532. Estes MK, Ball JM, Guerrero RA, et al. Norwalk virus vaccines: challenges and progress. J Infect Dis 2000; 181(suppl 2): S367–S373. Prasad BV, Hardy ME, Dokland T, et al. X-ray chystallographic of the Norwalk virus capsid. Science 1999; 286:287–290. Guerrero RA, Ball JM, Krater SS, et al. Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses. J Virol 2001; 75(20):9713–9722. Available at: http://www.ligocyte.com. 2007. Graham DY, Jiang X, Tanaka H, et al. Norwalk virus infection of volunteers: new insights based on improved assays. J Infect Dis 1994; 170(1):34–43.

Chapter 29: Virus-Like Particles as Vaccines and Vaccine Delivery Systems 51. Parashar UD, Gibson CJ, Bresse JS, et al. Rotavirus and severe childhood diarrhea. Emerg Infect Dis 2006; 12(2):304–307. 52. Cunliffe NA, Kilgore PE, Bresee JS, et al. Epidemiology of rotavirus diarrhoea in Africa: a review to assess the need for rotavirus immunization. Bull World Health Organ 1998; 76(5):525–537. 53. Widdowson MA, Meltzer MI, Zhang X, et al. Cost-effectiveness and potential impact of rotavirus vaccination in the United States. Pediatrics 2007; 119(4):684–697. 54. Estes MK, Kapikian AZ. Rotaviruses and Their Replication. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:1917–1974. 55. Jiang B, Gentsch JR, Glass RI, et al. The role of serum antibodies in the protection against rotavirus disease: an overview. Clin Infect Dis 2002; 34(10):1351–1361. 56. Vesikari T, Matson DO, Dennehy P, et al. Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 2006; 354(1):23–33. 57. Ruiz-Palacios GM, Perez-Schael I, Velazquez FR, et al. Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 2006; 354(1):11–22. 58. Buttery JP, Kirkwood C. Rotavirus vaccines in developed countries. Curr Opin Infect Dis 2007; 20(3):253–258. 59. Withdrawal of rotavirus vaccine recommendation. MMWR Recomm Rep 1999; 48(43):1007. 60. Crawford SE, Labbe M, Cohen J, et al. Characterization of viruslike particles produced by the expression of rotavirus capsid proteins in insect cells. J Virol 1994; 68(9):5945–5952. 61. Madore HP, Estes MK, Zarley CD, et al. Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine. Vaccine 1999; 17(19):2461–2471. 62. Crawford SE, Estes MK, Ciarlet M, et al. Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles. J Virol 1999; 73(6):4813–4822. 63. Fernandez FM, Conner ME, Hodgins DC, et al. Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines. Vaccine 1998; 16(5):507–516. 64. Coste A, Sirard J-C, Johansen K, et al. Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea. J Virol 2000; 74(19):8966–8971.

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65. Ciarlet M, Crawford SE, Barone C, et al. Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity. J Virol 1998; 72(11):9233–9246. 66. Azevedo MS, Yuan L, Iosef C, et al. Magnitude of serum and intestinal antibody responses induced by sequential replicating and nonreplicating rotavirus vaccines in gnotobiotic pigs and correlation with protection. Clin Diagn Lab Immunol 2004; 11(1): 12–20. 67. Conner ME, Estes MK. Virus-like particle (VLP) vaccines. In: Levine MM, Kaper JB, Rappuoli R, et al., eds. New Generation of Vaccines. New York: Marcel Dekker, Inc., 2004:283–293. 68. Dalba C, Bellier B, Kasahara N, et al. Replication-competent vectors and empty virus-like particles: new retroviral vector designs for cancer gene therapy or vaccines. Mol Ther 2007; 15(3): 457–466. 69. Ambuhl PM, Tissot AC, Fulurija A, et al. A vaccine for hypertension based on virus-like particles: preclinical efficacy and phase I safety and immunogenicity [see comment]. J Hypertens 2007; 25(1): 63–72. 70. Chackerian B, Lowy DR, Schiller JT, et al. Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies. J Clin Invest 2001; 108(3): 415–423. 71. Hirtz D, Thurman DJ, Gwinn-Hardy K, et al. How common are the ‘‘common’’ neurologic disorders? Neurology 2007; 68(5): 326–337. 72. Hock C, Konietzko U, Streffer JR, et al. Antibodies against [beta]amyloid slow cognitive decline in alzheimer’s disease. Neuron 2003; 38(4):547–554. 73. Gilman S, Koller M, Black RS, et al. Clinical effects of A{beta} immunization (AN1792) in patients with AD in an interrupted trial. Neurology 2005; 64(9):1553–1562. 74. Orgogozo JM, Gilman S, Dartigues JF, et al. Subacute meningoencephalitis in a subset of patients with AD after A{beta}42 immunization. Neurology 2003; 61(1):46–54. 75. Renner WA. Vaccine to treat nicotine addiction promotes 12 months continuous abstinence in subjects who achieve high antibody level. Available at: www.cytos.com. 2005.

30 Subunit Vaccines Produced Using Plant Biotechnology Qiang Chen, Hugh S. Mason, Tsafrir Mor, Alec Sutherland, Guy A. Cardineau, and Charles J. Arntzen The Biodesign Institute, Arizona State University, Tempe, Arizona, U.S.A.

Carol O. Tacket Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION A major focus of biotechnology is the improvement of human health around the globe. It is anticipated that the genomic revolution will greatly expand our knowledge of the molecular basis of many diseases and pathological states. Combining this knowledge with powerful screening techniques will be used in the development of safe and efficacious biologics and drugs for the prevention and treatment of disease. Unfortunately, the availability of these new biologics and drugs for use by all those who need them greatly depends on economic considerations such as the cost of their development, production, and delivery. Therefore, a major challenge of biotechnology is to translate clinical innovations to economically viable practice. The production of plant-derived vaccines for mucosal delivery is a step toward that goal.

WHY PRODUCTION AND DELIVERY OF VACCINES IN PLANTS MAKE SENSE Despite the public health success of current vaccines in controlling various infectious diseases, there are limitations in the current technology to develop vaccines against many emerging pathogens, especially those that have the ability to mutate rapidly and agents of bioterrorism. Production, manufacture, and delivery may be associated with high costs for vaccines against many of the pathogens for which vaccines are needed. Mucosal immune responses, characterized by production of secretory immunoglobulin A (sIgA) and the transport of these antibodies across the epithelium represent a first line of defense against pathogens that colonize and infect mucosal surfaces. As a result, stimulation of the mucosal immune system may be a particularly advantageous vaccination strategy. Further, mucosal vaccines that can be delivered by oral or nasal routes have the advantage of needle-free delivery, enhanced safety, and improved patient acceptance. Consequently, vaccine development focuses on finding new vaccines and improvements to traditional vaccines in terms of both source of immunogen and route of administration. Subunit vaccines that target stimulation of the mucosal immune system can potentially play an important role in vaccine development. To create these vaccines, a gene encoding a protective antigenic determinant from an infectious agent is

cloned, put under the control of an appropriate expression system, and transferred into a host organism. The transgenic host will then produce a ‘‘subunit’’ of the pathogen, a protein that cannot cause disease but can elicit a protective immune response. In most studies of the last 15 years, subunit vaccines have been purified from transgenic ‘‘production hosts,’’ for example, cultured yeast cells, and have been delivered via parenteral injection to immunize against a specific disease. To date, the only recombinant subunit vaccines licensed for use in humans are a yeast-derived hepatitis B vaccine, an Escherichia coli recombinant Lyme disease vaccine (no longer marketed), and human papilloma virus vaccine, which are delivered by intramuscular injection. While still in experimental stages of development, plantproduced, mucosally targeted subunit vaccines provide a strategy to improve the efficiency of antigen production and administration. Such plant-based vaccines merge innovations in medical science and plant biology. Work in this field was reported in the previous editions of this volume (1), and because of the subject’s interdisciplinary nature, importance, and appeal, plant-based vaccines continue to be periodically reviewed (2–12). In this chapter, we present an update on advances in the technology and, in particular, on recent phase I clinical trials conducted in the United States. We also address areas of research that require further attention if plant-based vaccines are to contribute to world health.

EXPRESSION OF ANTIGENIC PROTEINS IN PLANTS Plants can cost-effectively produce large amounts of functional proteins, free of animal pathogens, and production can be increased to agricultural scale. As a result, plant biotechnology approaches have emerged as a promising alternative to fermentation-based production systems for valuable pharmaceuticals. The ability of a plant to produce, correctly process, and assemble complex foreign proteins from a variety of organisms is well documented and is based on extensive research during the past 25 years in the realms of plant molecular biology, plant transformation, and plant virology. This work has not been confined to the developed world, as many scientists and public health authorities in developing counties have embraced the new technology for the possible future benefit of their people.

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Figure 1 A summary of the various strategies now in use to cause antigen expression and accumulation in plants. Antigen-encoding genes derived from pathogens are often resynthesized to optimize their expression in plant cells (codon usage, removal of cryptic introns, etc.). The most widely utilized option for expression of these genes has been to create transgenic plants (Option 1) in which either the chloroplast or the nuclear genome is stably transformed so that each cell in the resulting transgenic plant is a potential biomanufacturing center for the protein immunogens (2,3,7,8,11,34). In these plants, gene expression can either be constitutive or induced by exogenous agents. In Option 2, singlestranded RNA virus genomes are converted to DNA for manipulation in bacterial plasmids, a new gene encoding the antigen is inserted under a viral promoter control, and RNA is transcribed and used as the infectious agent to initiate viral genome amplification with the resulting concomitant production of the antigen (35–37). A variant of this approach is the engineering of the viral coat protein to cause expression of fused epitopes on the surface of the virus (37). In Option 3, viral RNA is also converted into a DNA sequence, engineered with a new gene, but the DNA is then moved into Agrobacterium; the bacterium is then infiltrated into leaves to express RNA from the DNA sequence and achieve ‘‘deconstructed virus’’ replication with the concomitant expression of the desired antigen (35,38–44).

The concept of using plant biotechnology to produce subunit vaccines has evolved over the last decade and a half of research. The initial focus was on the production of antigens in a food crop and then utilizing this ‘‘edible vaccine’’ as a means of oral immunization (1,3,6,7). The concept has been validated in preclinical and human clinical trials; both serum and mucosal antibody responses to food-delivered antigens have been documented in animals and humans (2,12–32). While these observations have stimulated a high level of academic interest, there has been no comparable corporate acceptance of ‘‘food-delivered vaccines’’ (2,11,33,34). Factors such as variability of antigen content in plant tissues, uncertainty of antigen stability during storage and transport, and a lack of clear path to licensure of plant-made vaccines have been perceived as major obstacles to introduction of a product. As a result, the focus of many plant-made vaccine efforts since about 2005 has moved to integrate plant-based antigen production with traditional downstream processing to yield purified immunogens that can be formulated and delivered in conventional vaccine systems. Since processing costs are directly related to antigen expression levels in cells or tissues, plant biotechnology efforts have largely concentrated on finding methods to enhance antigen accumulation. Chief among the emerging strategies to achieve high levels of antigen accumulation has been a switch from using transgenic approaches to transient, viral vector–based approaches. These changes in

strategies and techniques are discussed in the following sections. The various options for achieving antigen accumulation in plants are summarized in Figure 1.

PLANT-DERIVED VACCINES IN CLINICAL TRIALS The concept of producing subunit vaccine antigens in transgenic plants was first published in the scientific literature by describing the expression of hepatitis B surface antigen (HBsAg) in tobacco plants (13). Following the publication of their pioneering paper, the group headed by Arntzen and Mason continued to develop the concept of plant-based vaccines and reported their work in a succession of papers. The initial report focused on the expression and structure of the plant-produced HBsAg, which assembled into 22-nm virus-like particles (VLPs) similarly to the yeast-derived commercial vaccine antigen. Partially purified and concentrated tobacco-derived HBsAg was used in parenteral immunization experiments in mice, demonstrating its ability to invoke the expected B and T lymphocytic responses (14). To further prove that plant-derived HBsAg could stimulate mucosal immune responses following oral delivery, the group refocused their attention to expression in potato tuber. Surprisingly, the plant-derived material proved superior to the yeastderived antigen in both priming and boosting immune responses in mice (15). Success in these preclinical trials led to phase I studies with potatoes expressing HBsAg (16).

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To complement the efforts with HBV vaccine, the Arntzen and Mason group explored plant expression of other vaccine candidates—the heat-labile toxin B subunit (LT-B) of enterotoxigenic E. coli (ETEC) and the capsid protein of Norwalk virus (NVCP) (17–21). These antigens of two important enteric pathogens may represent examples of the ideal oral subunit vaccine candidate. Both are oligomers: LT-B configures as a pentamer, which has a high affinity to GM1 gangliosides present on mucosal cells (17), while NVCP can form VLPs (20). Furthermore, both have evolved to survive the extreme conditions of the stomach and infect [in the case of Norwalk virus (NV)] or colonize (in the case of E. coli) the gut epithelium. Another apparent advantage associated with plant expression of these proteins compared with HBsAg was accumulation to high levels in potato tuber. Significantly, both antigens assembled correctly into functional oligomers that could elicit oral immune responses in animals (18,20) and humans (19,21). The phase I clinical trial with potato tubers expressing LT-B not only provided the proof of concept that orally delivered plant-based vaccines could result in an immune response in humans but was also one of the first experiments of a pharmaceutical product derived from a transgenic plant conducted in humans. The clinical trials to date have examined both the safety and immunogenicity of plant-produced LT-B, NVCP, HBsAg, and rabies glycoprotein (12,16,19,21–23,34). In all these trials, individuals who consumed raw potato tubers or lettuce leaves containing approximately 0.3 to 1.0 mg of the antigens developed antibody responses. It is important to note that these antigens represent viral (NV, HBV, and rabies), bacterial (E. coli), enteric (NV and E. coli), as well as nonenteric (HBV and rabies) organisms. The titers of mucosal and systemic antibodies in some of the test subjects suggest that they would be protected from infection (19,21) and provide the justification for wider-scale clinical trials with these antigens.

PLANT-DERIVED VACCINES CAN PROVIDE PROTECTION AGAINST A PATHOGEN CHALLENGE The initial successes of early clinical trials encouraged other groups to explore the ability of plants to produce, fold, and assemble other vaccine candidates for the prevention of human and animal diseases. Various laboratories have reported efforts to produce transgenic plant-based vaccines for oral delivery to protect against human pathogens such as rabies (24), respiratory syncytial virus (RSV) (25), measles (45), rotavirus (46–48), and hepatitis B (22), and human cytomegalovirus (49,50), cholera (26,48,51,52), ETEC (27,48), and others have been reported. Plant-derived oral vaccines for veterinary use are aimed at foot and mouth disease virus (FMDV) (28– 30,53), swine-transmissible gastroenteritis virus (TGEV) (10,54–56), rabbit hemorrhagic disease virus (31), and Mannheimia haemolytica, the bacterial agent that causes shipping fever (32). While high titers of secretory and circulating antibodies following oral vaccination with plant-derived vaccines are important evidence of immunity, proof of efficacy requires that vaccination result in immune responses that are protective against a pathogen challenge. Veterinary vaccines provide an opportunity to assess the degree of immune protection directly. The series of papers published by the Borca group (28–30) serve as an excellent example of this methodological approach in a

veterinary context. Their first report described the use of a model plant system (Arabidopsis thaliana) for the expression of the VP1 protein of FMDV (28). Plant extracts containing VP1 provided full protection in mice after parenteral delivery and constituted the first demonstration of protection by a recombinant vaccine candidate produced in transgenic plants. Further studies using a larger number of mice immunized with extracts from transgenic potatoes (26) corroborated the initial work. They next expressed VP1 in alfalfa and delivered the transgenic plant material orally to mice. Despite low antigen expression, they achieved 70% protection against a virulent challenge after repeated oral boosting (30). The use of viral vectors has been used to produce a candidate vaccine to protect against a possible biothreat agent Yersinia pestis (39). Genes encoding the F1 and V antigens and the derived protein fusion F1-V were introduced into tobamovirus-based system vectors, which allowed very rapid and extremely high levels of expression (up to 1–2 g of antigen per kg of plant tissue). All three of the plant-derived purified antigens, administered subcutaneously to guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis.

FORMING MULTIVALENT AND MULTICOMPONENT VACCINES Vaccines designed to stimulate several facets of the immune system such as induction of strong humoral, mucosal, and cellular immune responses are highly desirable. Similarly, combination vaccines targeting multiple pathogens in one formulation are often desirable. Therefore, developing both multivalent and multicomponent plant-based vaccines would provide for both efficacious and cost-effective immunization strategies. Plants harboring transgenes encoding the antigens of several pathogens, either by direct transformation or through sexual crosses of individually transformed lines, or the blending of separately transformed plant tissues would easily fulfill this need. An alternative approach to achieve the same goal was taken by Yu and Langridge (48). They described a recombinant multicomponent vaccine based on cholera toxin (CT). They fused peptides containing important protective epitopes derived from two other enteric pathogens, ETEC, which causes bacterial traveler’s diarrhea, and rotavirus, which causes acute viral gastroenteritis, to the CT-A2 and CT-B subunits of CT, respectively. The two recombinant CT subunit fusions were expressed from a single bidirectional promoter, ensuring a coordinated expression pattern for the two gene fusions and potentially facilitating the assembly of the chimeric holotoxin. In this approach, CT provides a scaffold for presentation of the protective epitopes, acts as a mucosal targeting molecule without toxic effect due to use of the nontoxic CT-A2 and B subunits, and is itself a vaccine candidate. The recombinant protein represents a trivalent vaccine that can elicit significant mucosal and humoral responses against Vibrio cholerae, ETEC, and rotavirus. Mice, orally immunized with potatoes expressing these recombinant antigens, developed immune memory B cells as well as helper T-cell type 1 (Th1) responses, which are indicators of successful immunization. Further, pups of immunized dams were protected from challenge with rotavirus, with a significantly lower morbidity rate compared with controls. These results provide convincing evidence supporting a vaccine strategy employing chimeric proteins expressed in plants.

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PLANT VIRAL VECTORS AS ALTERNATIVE EXPRESSION SYSTEMS Low-level antigen expression in transgenic plants (0.01–2% of total soluble protein) has caused many groups of researchers to search for alternatives. Up to three doses of 100 g of raw potato tubers expressing LT-B were required in the clinical trials described above to elicit a significant immune response (19,21). It is likely that antigens with less immunogenic potential may require substantially larger doses to be effective. Even with more palatable alternatives to raw potatoes, such as tomatoes or tomato products, low levels of antigen expression could severely limit the utility of any vaccine product. As an alternative to transgenic plants, plant viral expression vectors have been constructed to allow efficient expression of recombinant proteins in plants (35–39). These vectors are of two types (Fig. 1). First, transgenic viruses incorporating a foreign gene express a recombinant product as the virus spreads throughout the plant. Alternatively, a deconstructed virus is designed in which the viral genome is delivered as DNA, which is transcribed in the plant to yield the viral RNA (which then amplifies in planta to yield high copy numbers). While plant virus–based vectors delivered as RNA have been useful in directing the expression of medically important transgenes, such as single-chain antibodies (57) and a hepatitis C virus hypervariable region 1/CT-B fusion (58), there may be certain insert size limitations. Alternatively, some plant virus vectors make use of coat-protein peptide fusions (36,59,60). Viral vectors based on tobamoviruses, such as tobacco mosaic virus, have received the most attention (37–39,58,60,61). Other plant virus vectors, which have been described, are based on geminiviruses (e.g., bean yellow dwarf virus) (35), potyviruses (e.g., plum poxvirus) (62), comoviruses (e.g., cowpea mosaic

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virus) (51,63,64), and bromoviruses (e.g., alfalfa mosaic virus) (65). The selection of antigens expressed by plant viral vectors includes bacterial pathogen-derived genes from Y. pestis (39), V. cholerae (48,58), Staphylococcus aureus (66), and Pseudomonas aeruginosa (61,67,68). An even larger selection of viral antigens such as norovirus (35,38), hepatitis C virus (58), RSV (65), rotavirus (69), human immunodeficiency virus (36,70,71), rabies (36,60), mink enteritis virus (64), canine parvovirus (64,72,73), FMDV (53), rabbit hemorrhagic disease virus (62), and hepatitis B core antigen (35) have been expressed via plant viral vectors. In most cases, viral vectors result in increased accumulation of the expressed recombinant antigens. Accumulation levels of the FMDV VP1 protein were low when the gene was expressed in stably transformed plants on the order of 0.005% to 0.01% total soluble protein (28,29,53) but were substantially higher when the antigen was expressed using a tobamovirus vector, approaching up to 150 mg/g fresh weight, or approximately 15% total protein (53). Interestingly, it is not uncommon to see similarly high levels of expression of stably integrated recombinant gene products in transgenic seeds without the use of viral vectors (10). In recent studies with deconstructed viral vectors delivered as DNA transcripts via Agrobacterium, which is transcribed to RNA viral transcripts that amplify in vivo, much higher levels of antigen have been achieved—allowing 0.5 to 2 g of subunit immunogen per kg of plant material. The success of viral expression vectors relies primarily on the fact that plant viruses are very efficient pathogens, employing very small genomes to infect their hosts. The use of Agrobacteriummediated transient antigen expression in tobacco has been ‘‘industrialized’’ to allow production of samples under Good Manufacturing Practice (GMP) guidelines (Fig. 2).

Figure 2 Virus infiltration into nontransgenic tobacco has been scaled to an industrial process. To meet GMP guidelines for antigen production, a defined tobacco seed bank has been established. In parallel, a master cell bank of Agrobacterium is developed and used for inoculum to deliver viral genomes (or deconstructed genomes) into plant cells. This is routinely achieved by submersion of tobacco plants in a bacterial suspension under vacuum. Infiltrated plants are returned to growth chambers to allow viral replication to proceed for periods of time up to about two weeks. Once optimal protein expression is achieved, plants are extracted and downstream protein purification is conducted under GMP protocols customary for other pharmaceutical manufacture. This slide summarizes processes that have been codeveloped by researchers at Arizona State University, MAPP Biopharmaceuticals, Inc. (San Diego, California, U.S.) and Kentucky Bioprocessing, LLC. (Owensboro, Kentucky, U.S.) and are being utilized to move a candidate norovirus vaccine and a monoclonal antibody–based microbicide into human clinical trials. Abbreviation: GMP, Good Manufacturing Practice.

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PLASTID TRANSFORMATION Over the past decade, data have been published on the transformation and expression of transgenes in plant chloroplasts, thereby taking advantage of the semiautonomous genetic machinery of these organelles (8,74–76). This approach to expression of biopharmaceuticals in crops was initiated primarily to meet perceived environmental transgene containment targets to ensure that genes are not lost to outcrossing. The concept relies on the strictly maternal inheritance of plastids in most species. Chloroplast transformation is an environmentally friendly way to engineer plants minimizing transfer to weeds or crops and decreasing the potential toxicity of pollen to insects (77). Other potential advantages of this system are high ploidy state, high transcription and translation rates, and the lack of gene silencing, all of which can contribute to high levels of foreign protein accumulation. While site-directed integration through homologous recombination appears to be a requirement, it might also provide more control of genetic engineering and increased uniformity of transgene expression (8,75,78,79). Additionally, one can make use of polycistronic operons, much like a bacterial system, to permit coordinated expression of multiple genes (79,80). Direct transformation of chloroplasts results in high levels of protein accumulation, up to several percent of total soluble protein, which is considerably more than that reported for other systems. In one case involving an operon from Bacillus thuringiensis, more than 40% was reported (79). Pharmaceutically important proteins expressed in plastids include human somatotropin (75), a biodegradable synthetic polymer (81), and also CT-B as a vaccine candidate (52). Plastid transformation has been extended to the experimental model plant A. thaliana (82) and two important solanaceous crop species, potato (83) and tomato (76). In relation to the latter two crops, transgene expression and recombinant protein accumulation were observed to occur in plastids that are specific for these two plants: potato tuber amyloplasts and tomato fruit chromoplasts. Unique advantages and disadvantages of plastid expression depend on the prokaryotic nature of the organelle, but so do its shortcomings. For example, Nglycosylation strictly depends on the endomembrane system. However, in the case of prokaryotic antigens or proteins and antigens that do not need to be glycosylated, plastids could possibly offer some distinct advantages.

OTHER TARGETS OF PLANT-EXPRESSED ANTIGENS Plant-expressed proteins have been proposed for other applications beyond prophylactic vaccines. For example, transgenic plants have been proposed for the production of autoantigens. Human autoantigens could be used to treat autoimmune diseases by inducing tolerance of the immune system rather than by stimulating it. An autoantigen implicated in diabetes, glutamic acid dehydrogenase (GAD), was produced in plants and fed to nude obese diabetic (NOD) mice, which have a particular susceptibility to the development of diabetes. This resulted in a reduction in pancreatic islet inflammation, an indication that immunotolerance had occurred (84). Arakawa et al. (85) used a similar approach feeding plant tissues expressing either proinsulin or a CT-B/proinsulin fusion to NOD mice, and they also observed a reduction in pancreatitis. This result suggested that they had immunotolerized the mice against this type of cytotoxic T cell–mediated autoimmune disease. In this case,

reduction of pancreatic inflammation coincided with increase in anti-insulin antibodies, mostly of the IgG1 isotype, leading to the conclusion that the cytotoxic T-cell response is suppressed. They were able to enhance this effect with the addition of a second antigen fusion, CT-B/GAD. It is interesting to note that the reduction in pancreatitis was considerably greater with the fusions than with the autoantigens alone, supporting targeting or adjuvant activity due to the CT-B component. We should also mention the differences in feeding protocols used in these two experiments. Ma et al. (84) fed very large amounts of recombinant GAD (1–1.5 mg per mouse per day) daily for four weeks, a more frequently used toleration protocol. Arakawa et al. (85), on the other hand, fed potato containing 20 mg of the CT-B-proinsulin fusion protein in five doses over a four-week period, which is almost identical to the feeding regimen they had reported to be effective in eliciting protective immune responses against foreign antigens (48). As Arakawa et al. suggested in their paper, the fusion to CT-B may facilitate the presentation of the antigen to the gut-associated lymphoid tissue to enhance the response. Transgenic plant-derived antigens have also been proposed for immunotherapy of malignant disease. These include, for example, plant-derived personalized human antibodies directed against non-Hodgkin’s lymphoma (NHL) (86) (see further discussion in sect. ‘‘Cancer Vaccines’’). Other groups have studied a plant-derived tumor-associated colorectal cancer antigen EpCAM that stimulated antibodies that inhibited the growth of colorectal cancer cells xenografted on nude mice (87). A third example is a plant-derived rabbit papilloma virus L1 antigen that stimulated protection against tumor challenge in rabbits (88). The control of overpopulations of wild mammalian species in a humane and effective manner through the use of immunocontraceptive plant-derived vaccines is another application of this technology (89,90). The approach is to express in plants a protein or a carrier protein harboring an antigenic epitope of an essential component of the mammalian reproductive system with the intention of inducing a humoral response following repetitive ingestion by the animal that results in sterility. The animal would remain sterile coincident with immune memory or until the next boosting vaccination. Antigenic targets reportedly under investigation include the gonadotropin releasing hormone and ZP3 from the zona pellucida of the mammalian ovulated egg (90,91). While effectiveness is still under study, this application is bound to meet considerable objections because of the danger that these broadly cross-reacting vaccines may be ingested by nontargeted mammalian species.

CANCER VACCINES An exciting recent development in the use of plant biotechnology for vaccine production has come from the manufacture of patient-specific vaccines against follicular B-cell lymphoma (86,92). Follicular lymphomas are a subtype of NHL, a malignant disease of the lymphatic system that is the seventh leading cause of cancer-related deaths in the United States (93). The administration of a tobacco-derived NHL vaccine in a human clinical trial resulted in immune responses in more than 70% of the patients, a majority of which showed a cellular response, suggesting that the vaccine will specifically direct the immune system to attack cancer cells. This was the first report on the clinical safety and immunogenicity of plant-made idiotypic

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Table 1 Plant-Derived Human Pharmaceuticals That Have Reached Clinical Trial Stage Organization

Product

Pharmaceutical target

Crop

Clinical trial stage

Arizona State University

Escherichia coli heat-liable toxin Hepatitis B virus surface antigen Norwalk virus capsid protein Interferon-a (Locteron) ScFvs

Traveler’s diseases Hepatitis B Norwalk virus Hepatitis C Non-Hodgkin’s lymphoma

Potato Tobacco Potato and tobacco Duckweeds Tobacco

Phase Phase Phase Phase Phase

I I I II I

Gastric lipase Lactoferrin sIgA (CaroRx) Glucocerebrosidase Insulin Hepatitis B virus surface antigen

Cystic fibrosis Gastrointestinal diseases Tooth decay Gaucher’s disease Diabetes Hepatitis B

Corn Corn Tobacco Carrot cell culture Safflower Lettuce

Phase Phase Phase Phase Phase Phase

II I II III I/II I

Rabies glycoprotein

Rabies virus

Spinach

Phase I

Biolex Therapeutics Large Scale Biology Corporation Meristem Therapeutics Planet Biotechnology Protalix Biotherapeutics SemBiosys Thomas Jefferson University

Source: From Refs. 2, 11, 34, and 94. Abbreviation: ScFvs, single-chain antibodies.

NHL antigens when administered subcutaneously in the absence of a universally used immunogenic carrier protein. In a directly relevant press release (92), Bayer AG announced in June of 2008 the opening of a production facility that will use tobacco to manufacture biopharmaceuticals and that the first proteins to be produced will be patient-specific biologics for follicular NHL therapy. This might be the first licensed plantmade vaccine for humans.

ISSUES AND CHALLENGES The remarkable growth of the field of plant-produced, orally delivered vaccines from its inception 20 years ago (13) to a research area involving many laboratories around the world has occurred despite initial skepticism. Plants have become a well-established platform for large-scale production of recombinant proteins, both therapeutic molecules and vaccine antigens (2,11,34,94); a summary of the candidate products that have advanced to human clinical testing is provided in Table 1. In the opinion of these authors, the next breakthroughs in public or commercial acceptance of plant-made vaccines are not going to be determined by technical obstacles related to plant molecular biology/virology. Instead, the major obstacles that must be overcome are related to regulatory requirements for vaccine introduction. Research to date, primarily in academia, has not focused extensively on adapting to GMP as required by the Food and Drug Administration in the United States and equivalent regulatory agencies in other parts of the world. Plant-made vaccines must meet all safety and efficacy requirements of vaccines made by any other process. It is likely that the plant growth and protein expression in planta can be established to meet regulatory requirements for licensure, since many candidate products have been allowed into clinical trials (Table 1), but ‘‘downstream protocols’’ to purify, concentrate, and formulate plant-made antigens into products that can be evaluated for safety, efficacy, and stability will take new skill sets. Extraction and processing procedures must meet quality control standards (95). The concept of a food plant-based vaccine presents itself to some immunologists as a contradiction in terms. They argue that the most common response to proteins presented in food is an induced state of specific unresponsiveness, called oral

tolerance (96–98). Tolerance plays an important role in the response of the host to environmental antigens and to resident microflora. At the very least, it would be a significant safety issue should a vaccine candidate induce long-term tolerance rather than a protective immune response. The issue is further complicated by reports in the literature of the use of comparable experimental approaches with similar constructs and feeding regimens to induce either immune responses or tolerance (48,85). Clearly, avoidance of tolerance to antigenic determinants of pathogens delivered via plant-based vaccines is critical. Dosing and scheduling regimens must be carefully studied to prevent such an outcome. Because of the high levels of antigen anticipated to be necessary for oral delivery and appropriate stimulation of the mucosa-associated lymphoid tissue, it is likely that an oral adjuvant will be required for optimal response. The inclusion of nontoxic mutant forms of CT or LT as part of a formulation may be one approach (99), while the identification of additional potential oral adjuvants should also be pursued. For example, plant products such as lectins and saponins are being explored for their oral adjuvanticity (70,100,101). Plants glycosylate proteins, and this can be advantageous (95). However, carbohydrate side chains, while added at the same sites in plants and animals, have slightly different structures. We do not know whether glycosylation structure will be important in the protective immune response. Another challenge is the low or variable expression levels of some antigens that affect feasibility of producing immunogenic dosing regimens. Careful identification and selection of the host plant combined with optimization of expression vectors and/or novel expression systems may provide a solution to low expression level. Further, compartmentalization of expression in discrete tissues may provide additional benefits with regard to controlled expression, level of protein antigen produced, and stability and may also affect processing technologies. Currently available food processing technologies may be employed to generate formulations that are not only more concentrated but also have delivery advantages. Even with the limitations of variable or modest antigen levels, prototypical plant-derived vaccines have been able to establish clinically relevant immune responses in adult human volunteers. Further refinement in plant-based vaccine development is an area of active investigation.

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66. Brennan FR, Jones TD, Longstaff M, et al. Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses. Vaccine 1999; 17: 1846–1857. 67. Brennan FR, Jones TD, Gilleland LB, et al. Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus. Microbiology 1999; 145:211–220. 68. Staczek J, Bendahmane M, Gilleland LB, et al. Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa. Vaccine 2000; 18:2266–2274. 69. O’Brien GJ, Bryant CJ, Voogd C, et al. Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into virus like particles. Virology 2000; 270:444–453. 70. McInerney TL, Brennan FR, Jones TD, et al. Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide. Vaccine 1999; 17: 1359–1368. 71. Marusic C, Rizza P, Lattanzi L, et al. Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. J Virol 2001; 75:8434–8439. 72. Gil F, Brun A, Wigdorovitz A, et al. High-yield expression of a viral peptide vaccine in transgenic plants. FEBS Lett 2001; 488:13–17. 73. Langeveld JP, Brennan FR, Martı´nez-Torrecuadrada JL, et al. Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus. Vaccine 2001; 19:3661–3670. 74. Heifetz PB. Genetic engineering of the chloroplast. Biochimie 2000; 82:655–666. 75. Staub JM, Garcia B, Graves J, et al. High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 2000; 18:333–338. 76. Ruf S, Hermann M, Berger IJ, et al. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol 2001; 19:870–875. 77. Daniell H, Khan MS, Allison L, et al. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 2002; 7:84–91. 78. McBride KE, Svab Z, Schaaf DJ, et al. Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco. Biotechnology (N Y) 1995; 13:362–365. 79. De Cosa B, Moar W, Lee SB, et al. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 2001; 19:71–74. 80. Staub JM, Maliga P. Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J 1995; 7:845–848. 81. Guda C, Lee SB, Daniell H, et al. Stable expression of a biodegradable protein-based polymer in tobacco protoplasts. Plant Cell Rep 2000; 19:257–262. 82. Sikdar SR, Serino G, Chaudhuri S, et al. Plastid transformation in Arabidopsis thaliana. Plant Cell Rep 1998; 18:20–24. 83. Sidorov VA, Kasten D, Pang SZ, et al. Technical advance: stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J 1999; 19:209–216. 84. Ma SW, Zhao DL, Yin ZQ, et al. Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nat Med 1997; 3:793–796. 85. Arakawa T, Yu J, Chong DK, et al. A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat Biotechnol 1998; 16:934–938. 86. McCormick AA, Reddy S, Reinl SJ, et al. Plant-produced idiotype vaccines for the treatment of non-Hodgkin’s lymphoma: safety and immunogenicity in a phase I clinical study. Proc Natl Acad Sci U S A 2008; 105(29):10131–10136.

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31 Lipopeptide-Based Vaccines David C. Jackson, Brendon Y. Chua, and Lorena E. Brown Department of Microbiology & Immunology, The University of Melbourne, Parkville, Victoria, Australia

Peter Moyle and Istvan Toth School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia

INTRODUCTION The move to rational design of vaccines can, at least in part, be attributed to the demands of authorities for compliance with a host of regulatory requirements. The ability to rationally design the next generation of vaccines can be attributed to our increasing understanding of how the immune system recognizes antigen and how it then responds to it. The most relevant fields of discovery that have enabled this are (i) an awareness of the role that short peptide sequences, the epitopes, play in immune recognition; (ii) an appreciation that among the first cells to encounter pathogens are cells of the innate immune system of which the dendritic cell (DC) is of particular importance; and (iii) an understanding of the different types of immune responses that are associated with recovery from infection. Information from all of these areas provides us with insights as to the form of antigen and its method of delivery that will lead to an appropriate immune response. As details of the rules governing antigen recognition and stimulation of the immune system have emerged, a rapidly increasing literature and an escalation in the inventory of vaccine technologies have followed, promising new and improved approaches to immunization. Among these technologies is the utilization of synthetic peptides, which have been the tools of immunologists for many years. Although the use of peptides has been largely restricted to their application in basic research, the prospect of making totally synthetic vaccines has been a recurring theme. The concept of using synthetic peptides as a basis for vaccine design is simple; if the epitope that is recognized by an antibody or a T cell or some other effector of the immune system is known, then a vaccine can be designed around that epitope. A simple idea perhaps but the method of delivery of the epitope(s) is of paramount importance and our ignorance of that has delayed the realization of totally synthetic vaccines. Once the basis of recognition of short peptides by receptors on T lymphocytes was understood, peptide epitopes became an obvious choice for inducing T-cell immunity (1–3). Furthermore, because some antigens possess B-cell epitopes that can be mimicked by synthetic peptides, epitope-based vaccine candidates were also investigated for their ability to induce antibodybased immunity (4–6). Short peptides, however, induce T- and Bcell responses only when administered with potent adjuvants (7,8). We now understand that this is because simple peptides lack features that are an inherent property of many proteins or

other components of invading pathogens that the immune system has evolved to recognize as foreign and dangerous. With this knowledge we are now in a position to apply some of what we know about the ligands that provide these ‘‘danger signals’’ that are relayed by receptors, such as the Toll-like receptors (TLRs) present on antigen-presenting cells (APCs), and incorporate these ligands into new candidate vaccines. We are also beginning to apply what we know about the transport mechanisms operating in cells to transport vaccine cargos into the correct compartments for appropriate antigen processing. Apart from an appreciation of the importance that short peptide sequences play in the induction of immunity, a number of technical advances have contributed to the feasibility of designing totally synthetic vaccines. Long sequences of amino acids can now be synthesized with confidence using modern synthesizers, including those that make use of microwave technology to facilitate coupling reactions (9). These instruments now make the synthesis of small proteins (
60 amino acids) feasible. Chemoselective ligation procedures (10–21) allow synthetic peptide modules to be ligated, producing multimeric immunogens. The ability to assemble multivalent antigens allows us to incorporate different epitopes from multiple serotypes of pathogens as well as series of epitopes that cover the polymorphic class I and class II molecules of the major histocompatibility complexes (MHCs) within the target species. Advocates of peptide or epitope-based vaccines have been pursuing their trade for three decades, starting perhaps with the encouraging and seminal study of Langbeheim et al. (22) in 1976, where antibodies raised against MS-2 coliphage synthetic fragments were able to neutralize the virus. Since then, however, the poor immunogenicity of peptides, the difficulty in raising antibody against native antigens using epitope approaches and the problems of multivalency, have produced a general air of disenchantment in the minds of many vaccinologists and also of those in control of strategic policy within pharmaceutical companies when it came to considering totally synthetic vaccine strategies. Now, with these new technical advances and insights into immune mechanisms, there has been a paradigm shift, resulting in a major revision in the way in which epitope-based vaccines are viewed. The recent design of successful peptide-based vaccine candidates against infectious diseases including viruses (23–27), bacteria (27–30), parasites (31–34), as well as tumors (35–38) and self-hormones,

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which allow modulation of physiological processes (20,27,39), supports these views. While the early promise of totally synthetic epitope-based vaccines was premature, with the wisdom of hindsight we can now explain early failures and the benefit of advances in our knowledge of innate immunity and the role played by peptides in immune recognition has led us to a phase of discovery and design that has finally resulted in attention being paid by Big Pharma.

ADVANTAGES, LIMITATIONS, AND SOLUTIONS The varied and different approaches to vaccine design are not mutually exclusive. In those situations where we do not know which antigen(s) is necessary to induce immunity, a whole organism approach is warranted. In other cases where it is known which individual protein needs to be targeted a recombinant protein approach makes sense. In those cases where individual epitopes have been identified and where whole proteins may contain deleterious sequences (40–42), an epitopebased vaccine may be the answer. Some of the advantages, potential and realized, of peptidebased vaccines include the following: l

l

l

l

l

l

l

l

l

No need for infectious material; apart from safety issues in growing pathogens, many are difficult or impossible to culture. No risk of reversion or formation of adverse virulent reassortants, a potential limitation of live attenuated vaccine preparations. No possibility for genetic integration, a problem facing DNA vaccination. Deleterious sequences such as oncogenic sequences within Epstein-Barr virus or sequences that are implicated in autoimmune phenomena can be omitted. Immunogenicity, stability, and solubility can be improved or modulated by the simple introduction of lipid, carbohydrate, and phosphate groups. Robust and well-established analytical techniques such as high performance liquid chromatography and mass spectrometry can be used for quality assurance. Production of peptides on a large scale can be carried out economically. Peptides can be stored freeze-dried avoiding the need to maintain a ‘‘cold chain’’ during storage, transport, and distribution. Multiple antigenic epitopes from the same pathogen or numerous antigenic determinants from different pathogens can be assembled into the one vaccine.

Another advantage that synthetic vaccines possess is the ability to assemble them using unusual geometries, which appear to be attractive to DCs (43). A number of branched structures, not found in nature, have been assembled that possess an ability to provoke useful immune responses (16,19,43–46). Despite this list of advantages and before the successful implementation of epitope-based vaccines can be achieved, a number of issues do need to be addressed when designing this type of vaccine.

1.

The immunogenicity of peptides is inherently low, and they need to be administered in adjuvant to obtain useful antibody titers and/or levels of cytotoxic T lymphocyte

2.

(CTL) activity. Poor immunogenicity is of course a feature of many soluble protein-based antigens that also have to be administered with exogenous adjuvants. The most potent of these often contain pathogen-derived components [e.g., heat-killed mycobacteria present in complete Freund’s adjuvant (CFA) or the synthetic muramyl dipeptide that is obtained from bacterial peptidoglycan] but they are not always suitable for use in humans because of their inherent toxicity (47,48). In the case of synthetic peptidebased vaccines, however, a number of solutions including the incorporation of nontoxic and self-adjuvanting lipid moieties can be applied, which will be discussed in detail below. The specificity of antibodies elicited by epitope-based vaccines is often inappropriate; many anti-peptide antibodies are unable to bind to the native protein antigen. A seemingly constant argument leveled against peptide-based immunogens and vaccines centers around the ‘‘conformational’’ versus ‘‘linear’’ epitope issue. This relates to the fact that many attempts to produce antibodies using peptide-based immunogens elicit antibodies capable of binding to the immunizing peptide but are incapable of binding to the native protein from which the peptide epitope is derived. In those cases where the amino acid sequence of the B-cell epitope is known, peptides representing them have little secondary structure and they rarely adopt the conformation exhibited in the native protein. Antibody produced in response to such peptides, therefore, may not be specific for the original and structurally different native antigen. There is no doubt that this has been the case in many studies; but as our skill in assembling peptides and constraining their conformation increases along with information on the three-dimensional (3-D) structure of antigens, we are seeing that respectable and often startlingly high-titer antibodies are being obtained using totally synthetic vaccines. An example of a successful approach to providing the appropriate conformation into a synthetic peptide for the purposes of constructing a vaccine comes from the work of Good’s group on the M protein of group A Streptococcus (GAS) (49,50). The M protein is a coiled-coil a-helical surface protein and induces antibody that is able to opsonize bacteria and protect animals from infection (51). The conserved region of the M protein that, when mapped with antibodies from the sera of most adults living in areas of high GAS exposure, was found to contain a protective B-cell epitope (52). Synthetic peptides representing this sequence did not, however, possess the necessary helical conformation to allow them to function as epitopes but when flanked by helix promoting sequences from the yeast transcription factor GCN4, the chimeric peptides were shown to possess a-helical conformation (50,52). Antibodies elicited by a vaccine candidate based on these chimeric structures were opsonic and protective against GAS infection (53). These studies elegantly highlight the fact that in at least some cases a knowledge of the 3-D structure of the native antigen is required before antibody-inducing epitope-based vaccines can be effective. Needless to say in the case of short peptide epitopes such as peptide hormones [e.g., luteinizing hormone releasing hormone (LHRH) or gonadotrophin releasing hormone (GnRH), which is only 10 amino acid residues in length], there is

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sufficient information built into the native sequence that the correct conformation is promptly attained and antibodies readily elicited (27,54–56). In the case of T-cell epitopes, which are not recognized in their native structure as in the intact protein but only when enzymatically processed and loaded onto MHC molecules, conformation is not an issue and T-cell epitope-based vaccines can be readily synthesized. The T- and B-cell epitopes defined in a single host of a particular MHC type may be inadequate for eliciting immunity in outbred populations with polymorphic MHC molecules. In addition, many diseases are caused by organisms where the target antigens show a high degree of genetic variation, creating multiple serologically distinct variants. An important consideration therefore is to provide sufficient numbers of different epitopes. Protein antigens contain multiple B-cell epitopes and often also possess epitopes that will be recognized by helper T cells, providing the help that is necessary for antibody production. Likewise a pathogen may have a range of different CD8þ cytotoxic T-cell epitopes that can be used as vaccine targets despite the fact that only one or two may dominate in the response to the whole organism. In other words, whole proteins or whole pathogens usually contain all of the information that is needed to produce a poly-specific immune response. Thus, for epitope-based vaccines to elicit such responses, all this information should ideally be incorporated into the vaccine. A number of methods for doing this are available and include the multiple antigenic peptide (MAP) approach of Tam and colleagues (46,57) involving assembly of multiple peptides onto a branched oligolysine support, and the synthesis of peptides on cross-linked acrylamide supports where the cross-links are cleaved on exposure to trifluoroacetic acid, resulting in a long single chain polyamide to which multiple copies of the peptide are attached (58).

There are, however, limitations to the degree of purity of the materials that are achieved with these approaches. As the valency of the vaccine candidate increases, the heterogeneity of the product also increases, making quality assurance difficult. Although the elegant chemical ligation approaches described by Rose (59) and Tam (60,61) addressed these issues providing greater flexibility by permitting the conjugation of different purified peptides onto a template support, the number of different peptide epitopes that can be incorporated into these structures is still limited by the number of orthogonal chemistries available. A number of approaches permit peptides to be synthesized, purified, and then assembled into polymers. Using the technique of free radical–induced polymerization of peptides (16,17,62), very large (>600,000 Da) molecular species can be assembled using virtually any number of the same or different epitopes. The method allows purification of the individual determinants prior to polymerization and thereby avoids errors inherent in long sequential syntheses. The approach has been successfully used to design and assemble GAS vaccines containing multiple epitopic variants present in different isolates (19) and also hepatitis C virus vaccines based on multiple B-cell epitopes in the form of a library covering possible variants of the hypervariable region of the surface antigen of the virus (63) (Fig. 1).

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Polyvalent, self-adjuvanting vaccines have also been produced using the multiple-antigen lipophilic adjuvant carrier (MALAC) system (Fig. 2) (64–67), which utilizes site-specific conjugation of purified peptide epitopes into a lipoaminoacidbased scaffold. Using this approach, GAS lipopeptide vaccines containing different peptide epitopes have been synthesized in good yield and purity and been demonstrated to elicit hightiter antigen-specific antibodies (67).

EPITOPE IDENTIFICATION Before a synthetic vaccine can be designed, appropriate epitopes must be identified. Epitope identification is and has been a continuing endeavor of immunology, and as a consequence a large number of different methods have been applied to the identification of these, the smallest, elements that are recognized by antibodies and T-cell receptors. The suite of methodologies available range from simple ELISA and even Ouchterlony-related methods to the use of panels of synthetic peptides representing complete sequences of antigens and the sophisticated techniques of electron microscopy, NMR, X-ray crystallography, and the use of mass spectrometry to characterize protease or chemically derived fragments. Many of these disparate techniques can be mixed and matched to identify epitopes in almost any antigen. The plethora of protocols available reflects the number of solutions available and invites an eclectic approach to the solution of epitope mapping. Epitope mapping has often been done using monoclonal antibodies (MAbs), and although there is no doubt that MAbs provide exquisite specificity, the case should be made that the antibody response is polyclonal and more attention should perhaps be paid to polyclonal antibodies. Furthermore, antisera exhibit properties not always exhibited by MAbs. These include the ability to refold antigens through concerted and multiple antibody-binding events; some isolated subunits of antigens with quaternary structure are not bound by MAbs but are recognized by antisera. Immunoglobulin (Ig) purified from such antisera can be useful where MAbs are not. Furthermore, it is antisera from patients recovering from disease that may provide very useful information concerning identification of biologically important epitopes. If patients have their serum antibodies capable of neutralizing a virus, then information about those epitopes is present in the binding sites of those antibodies. Ig isolated from individuals with past or current infection can therefore be used to ‘‘mine’’ a panel of peptidebased epitopes that represents the complete amino acid sequence of any protein to discover epitopes of significance. Peptides that are bound by Ab can then be isolated and identified by a process of ‘‘epitope extraction.’’ This general approach to epitope identification was pioneered by Suckau et al. (68) and more recently utilized by others (69,70). The method has lately been applied to the identification of novel, and potentially neutralizing, epitopes of hepatitis C virus (71). In the case of T cells, the epitopes that they recognize can now be quickly and easily identified using peripheral blood mononuclear cells (72,73) or with techniques using whole blood–based assays (74,75). Longitudinal studies of immune responses in various disease states can provide insights into the relevant CD8þ or CD4þ T-cell responses that correlate with recovery, which can be further validated in models of the target disease using mice that are transgenic for the MHC alleles expressed by humans. In either case, lymphocytes are

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Figure 1 Scheme for the preparation of synthetic peptide–based polymers. Peptides are assembled on solid phase supports and then acylated at the N-terminus with acryloyl chloride. Following removal of the peptide from the support and concomitant removal of the side chain protecting groups, the peptide epitopes are purified and polymerized by exposure to free radical. The molecular models at the right represent, to the same scale, an IgG Fab fragment (*50,000 Da) and a portion of a polymer formed by the free radical-induced polymerization of multiple peptide epitopes. R¼H or R¼CN.

stimulated with a series of peptides from the target protein. Such assays are very sensitive and, by judicious use of 18residue and approximately 9-residue peptides, allow the identification of CD4þ and CD8þ T-cell epitopes. Ab and T-cell epitopes identified by each of these methods now become candidates for incorporation into a single synthetic structure for inducing broad-based immunity. This strategy has advantages over using a recombinant protein because epitopes from multiple and relevant proteins can be included and deleterious sequences can be excluded. Caution, however, must be taken in the selection of T-cell epitopes especially when the disease pathology is caused by inappropriate T-cell activity. The topic of epitope identification has been the subject of many reports in the scientific literature and is the subject of the laboratory handbook Epitope Mapping Protocols (76).

STRUCTURES OF LIPOPEPTIDE-BASED VACCINES Representative structures of bacterial-derived or synthetic lipid moieties that potentially have the capacity to provide an adjuvanting effect for peptide epitopes, including points of modification, are shown in Figure 3. Sites of modification that have been investigated include the (i) the N-linked fatty acids (R1), (ii) the O-linked fatty acids (R2 and R3), (iii) the chirality

of the glycerol backbone, (iv) cysteine residue chirality, (v) sulphur atom substitutions within the cysteine residue (R4), and (vi) variations in the carboxyl-terminal peptide sequence (R5). Changes to the O-linked fatty acids incorporated within the lipid moiety appear to have the greatest effect on adjuvant activity. For both Pam2- and Pam3-Cys analogues, the presence of O-linked palmitoyl groups (C16) provides the best adjuvant activity, with shorter (
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Figure 2 Scheme for the synthesis of the multiple antigen lipophilic adjuvant carrier system. Multiple peptide antigens, synthesized as carboxyl-terminal thioester peptides, are conjugated to an amino-terminal cysteine peptide containing the lipidic adjuvant. Ligation reactions are performed, followed by removal of cysteine protecting groups, until the lipopeptide vaccine of interest is synthesized. Abbreviations: PG, protecting group. MALAC, multiple antigen lipophilic adjuvant carrier.

oligolysine. The LCP system has been used to produce vaccines against various microorganisms including Streptococcus pyogenes of the GAS (94–97), Chlamydia trachomatis (98), and human papillomavirus type-16 (99). An alternate

strategy to the LCP system is the MALAC system (94,95) mentioned above (Fig. 2), which provides a multiepitope vaccine-containing lipid that has been used to assemble GAS lipopeptide vaccines candidates (95).

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Name Triacylated P3C-SSNA P3C-SK4 P3C-S P3Adh-SK4 Diacylated MALP-2 MALP-2- SK4 P2C-SK4 PC(P)-SK4 Monoacylated PC-SK4

R1 fatty acid

R2 fatty acid

R3 fatty acid

R4 group

R5 peptide

References

Pam Pam Pam Pam

Pam Pam Pam Pam

Pam Pam Pam Pam

S S S CH2

SSNA SKKKK S SKKKK

(77) (78–83) (77) (79)

H H H Pam

Pam Pam Pam H

Pam Pam Pam Pam

S S S S

GNNDESNISFKEK GNNDESNISFKEKSKKKK SKKKK SKKKK

(84) (82,84) (80–82,84) (80,85)

Pam

H

H

S

SKKKK

(80,81)

Figure 3 Structural formula for various lipopeptide derivatives with the main points of variation indicated. The peptide sequence at R5 is described using single-letter notation.

LIPID-MEDIATED DELIVERY AND ADJUVANTING EFFECTS Reports that the immunogenicity of peptides could be improved by the incorporation of lipids began to appear in the 1980s heralding the development of self-adjuvanting synthetic vaccines (reviewed in Refs. 100–103). Hopp (104) demonstrated that improved immunogenicity resulted following acylation of a peptide derived from hepatitis B surface antigen with two palmitic acid residues. Subsequently tripalmitoyl-Sglyceryl-cysteine (Pam3Cys) (Fig. 3) was covalently attached to a CD8þ T-cell epitope derived from influenza virus and shown (105–107) to be capable of inducing T cells. Pam3Cys is a synthetic version of the lipid component of Braun’s lipoprotein (108), which is a constituent of the cell wall of gramnegative bacteria. Its derivatives include Pam2Cys, which occurs naturally in the Mycoplasma-derived macrophageactivating lipopeptide-2 or MALP-2 (109). It is a simple matter to speculate that the immune systems of vertebrates have evolved to recognize the signatures of microorganisms including these lipid structures, and, in fact, endogenous receptors present on DCs demonstrate specificity for such compounds. It is clear that the adjuvanting properties of such lipids are due to their ability to be recognized by Toll-like and other receptors on APCs.

Whether antibody or T cells are required to mediate the immune response that is sought, it is essential that DCs are involved. These cells capture and process antigen and then present the resulting antigenic peptides to specific T cells. DCs express class I and class II MHC molecules on their surface and are therefore able to present antigen to CD8þ or CD4þ T cells. In fact, DCs are the only APC that can efficiently prime naive T cells (110,111) and are, as a consequence, the cell that vaccines need to target. DCs are widely distributed throughout the body including the skin. They patrol most tissues and have on their surface and internal membranes an array of receptors that have evolved to recognize many of the surface features of pathogens including the lipids and carbohydrates of structural lipoproteins and glycoproteins or the pathogen genomes. Engagement by a particular DC surface receptor can allow pathogenic material to be transported inside the cell and processed for presentation to cells of the adaptive immune system. The result of this concert of events will be a specific cellular or antibody response to particular epitopes of the invading pathogen. If ways can be found to specifically target DCs, through their specific pathogen-recognizing receptors, then the efficacy and potency of vaccines are improved. DCs have been called Nature’s adjuvants and in fact the adjuvants that have been used in the laboratory and clinic function in their various ways by directly or indirectly stimulating DCs.

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Figure 4 Schematic of simple epitope-based lipopeptide vaccines. (A) The vaccine is a simple and single target (cytotoxic T lymphocyte) epitope with lipid attached to the N-terminus. (B) The vaccine comprises a helper T-cell epitope and a target epitope, which can either be an epitope that induces antibody or one that induces CD8þ T cells, the lipid is attached to the N-terminus of the peptide sequence comprising both epitopes that is assembled as a single contiguous sequence. (C) The vaccine again comprises helper T-cell epitope and a target epitope, but in this case the lipid moiety is attached as a branch between the two peptide epitopes. (D) Photograph of left, a solution of branched lipopeptide, and right, a solution of linear lipopeptide showing improved solubility of the branched configuration. In the case of antibody-inducing vaccine candidates, the helper T-cell epitope is necessary for T-dependent antibody induction and B-cell differentiation to produce various antibody isotypes; in the case of CD8þ T-cell induction, the helper T-cell epitope is necessary for establishment of CD8þ T-cell memory.

MECHANISMS OF ACTION The TLRs comprise a family of receptors present on DCs that recognize molecular patterns unique to microorganisms and alert the host to invasion by pathogens. Their function and mechanism of action (112) provide a link to understanding the mechanisms of action of some lipopeptide vaccines. Different members of the TLR family recognize particular microbial components: TLR4 recognizes bacterial lipopolysaccharide, TLR5 recognizes bacterial flagellin, TLR9 recognizes unmethylated CpG motifs of bacterial DNA, TLR3 recognizes doublestranded RNA, and TLR7 recognizes single-stranded viral RNA (for a review see Ref. 113). TLR2 is essential for the recognition of bacterial lipoprotein and lipopeptides (114); some cells recognize microbial lipopeptides and lipoproteins through the formation of heterodimeric complexes of TLR2 with other TLRs. TLR6-deficient mice show an impaired response to mycoplasmal lipopeptides that are diacylated, whereas TLR1-deficient mice are defective in their response to bacterial lipopeptides that are triacylated. TLR2-deficient mice do not show any inflammatory response to either type of lipopeptide. It appears then that TLR1 and TLR6 are involved in the discrimination of differences between Pam3Cys and Pam2Cys through interaction with TLR2, although both the lipid and the N-terminal amino acid

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sequences of lipoproteins may also contribute to the specificity of recognition by TLR2 heterodimers (84). Within Pam3Cys, it is the two ester-bound acyl chains rather than the amide-bound fatty acid molecule that provide the major contribution to TLR2-dependent cellular recognition (86). Apart from providing a means of targeting DCs by incorporating Pam3Cys or Pam2Cys into vaccines, the endocytic nature of TLR2 (115) also provides a means of delivering peptide cargo into the APC. Furthermore, interaction of TLR2 with its ligand triggers a signaling cascade that results in the translocation of the transcription factor nuclear factor-kappaB (NF-kB) to the nucleus and the subsequent switching on of NF-kB-dependent genes that are responsible for the phenotypic and functional changes that accompany DC maturation. Mature DCs are capable of efficient antigen presentation of peptides on MHC molecules and migrate to lymph nodes draining the site of antigen uptake where they are exposed to naive T cells. With the increased level of costimulatory molecules on their surface, resulting from the maturation process, the mature DCs are capable of activating those naive T cells with receptors specific for the peptide MHC that they are presenting. Pam3Cys has been shown to activate or repress an array of at least 140 genes partly involved in signal transduction and regulation of the immune response (116). We (27,87,89) have shown that branched Pam2Cys-containing lipopeptides trigger the readout of NF-kB-dependent genes in a TLR2-dependent manner resulting in the induction of strong CD8þ T cell and antibody responses. By exploiting the properties of TLR2-based receptors present on DCs, lipopeptide vaccine candidates exert their self-adjuvanting activity by mimicking bacterial lipopeptides or lipoproteins using the mechanisms that the host has evolved to initiate strong immune responses to counter bacterial invasion. Less obvious is how lipopeptide vaccine candidates with one or two isolated palmitic acid residues, as opposed to when in the context of an acylated Cys, exert their biological effect because these lipid components are only distantly related to those derived from bacterial lipopeptides and proteins. Early studies using fluorescently labeled lipopeptides indicated that these bound rapidly to the surface of cells during incubation in vitro (117) and demonstrated destabilizing effects on model lipid membranes (118). It was suggested that the lipopeptide anchored in the membrane allowed peptide cargo direct access to the cytoplasm. This provided a potential explanation as to how CD8þ T-cell epitopes could access the class I processing pathway. The fact that a bi-palmitoylated lipopeptide consisting of a helper T-cell epitope covalently linked to an influenza virus CTL epitope exhibited virtually no binding to purified class I molecules (119) suggested that such lipopeptides need to be internalized for processing. To follow class I processing pathways in human DC, Andrieu et al. (120) tracked a lipopeptide comprising an HIV epitope covalently attached to palmitoyllysine. The lipopeptide and its parent peptide were fluorescently labeled and their entry into immature monocyte-derived human DC was examined by confocal microscopy. It was found that, rather than directly transporting across the plasma membrane, the lipid moiety induced energy-dependent endocytosis. A more recent finding by Zhu et al. (121) is that lipopeptide epitopes, extended by a single N-epsilon-palmitoyllysine moiety, exhibit increased uptake by and maturation of DC through a TLR2-dependent pathway, a finding that perhaps extends receptor-mediated mechanisms to simple lipid structures. However, a lipopeptide comprised T-cell epitopes from

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influenza virus and two palmitic acid groups attached to the peptides through a lysine residue, failed to signal through TLR2, mature DC, or trigger IL-12 secretion at levels where Pam2Cysand Pam3Cys-containing lipopeptides were highly active (89). Perhaps increased stability and persistence of the MHC-peptide complex contributes to enhancing lipopeptide immunogenicity as found with a palmitoylated version of a vaccine candidate for human papillomavirus in HLA-A2 transgenic mice (122). The innate and adaptive immune systems provide multiple points at which lipids can have an effect, and the interplay between the two systems often enables downstream amplification effects; Pam3Cys- and Pam2Cys-based lipid moieties, for example, cause macrophages and monocytes to release proinflammatory cytokines as well as chemokines that attract neutrophils and other white cells (123–125). Expression of TLRs on tissues other than DCs provides opportunities for input of additional stimuli into the immune system; B cells present in the nasal-associated lymphoid tissues constitutively express TLR2 and engagement of MALP-2 causes the upregulation of class II molecules and the expression of costimulatory molecules. Such stimulation could improve these cells’ antigenpresenting properties as well as making them more accessible to the help provided by their interaction with CD4þ T cells. Many of these properties are retained by lipid-conjugated vaccine epitopes and antigens including lipopeptides containing a single palmitoyl lysine residue (126). It is clear that the ability of some lipopeptide-based vaccines to specifically target DCs and to be efficiently transported across the cell membrane through interaction with surface receptors ensures efficient antigen uptake by DCs. The ensuing events of cellular maturation including expression of class II and costimulatory molecules that can also occur clearly improve the efficacy of lipopeptide-based vaccines. The fact that the underlying mechanisms of action are understood also admits them into the repertoire of rationally designed vaccines.

CODELIVERY OF LIPOPEPTIDES WITH ANTIGEN The immunostimulatory properties of lipopeptides have also been exploited by admixing with antigens in much the same way that adjuvants are admixed with antigen prior to administration. Such lipopeptides generally have little or no intrinsic immunogenicity. The water-soluble lipohexapeptide Pam3CysSer-(Lys)4 was shown to improve the antibody response to a variety of antigens (127–129) and has also been shown to promote type 1 cytokine responses (130). In a direct comparison of different adjuvants, Pam3CysSer-(Lys)4 either administered with malaria CTL epitope or when covalently attached to it was found to be superior to other formulations (131). As a consequence of the finding that simple lipopeptide-based adjuvants can be administered with antigen to enhance ensuing immune responses, a great deal of effort has been applied to optimizing these structures (78,132–135) for maximum biological function. Recent work in this area has focused on the use of a synthetic version of MALP-2 [which contains Pam2Cys as the lipid component) as a co-delivered adjuvant for both mucosal and systemic delivery (136) and has been reported to enhance the antibody response to the Tat (137) and matrix (138)] proteins of HIV. In addition, intranasal co-inoculation of MALP-2 with live-attenuated measles vaccine virus lead to the induction of higher titers of neutralizing antibodies leading to protective immunity in cotton rats (139). Some studies have shown that

covalent attachment of the lipid to the peptide immunogen is more efficient for a given dose of peptide (56,132), but the codelivery approach does have the advantage of utility with vaccine candidates that are not readily synthesized. The strong immunostimulatory effects of lipopolysaccharide have also attracted immunologists for many years but its toxic effects have prevented its adoption into the field of vaccinology. Attempts to identify an adjuvanting, nontoxic component of LPS resulted in the development of monophosphoryl lipid (MPL) A. MPL is a mixture of six glycolipids and is obtained through sequential acid-base hydrolyses of bacteriaderived LPS (140). MPL A has been demonstrated to possess many of the adjuvant properties of LPS with fewer side effects (141). By 2005, over 273,000 MPL doses had been administered in clinical trials, with a high degree of safety and superior adjuvant activity compared with alum (141). These results have led to GlaxoSmithKline’s MPL-adjuvanted hepatitis B vaccine, Fendrix, receiving marketing approval in Europe. Synthetic analogues of LPS have also been synthesized (142,143), which tend to be single chemical entities as opposed to mixtures of compounds. The majority of MPL studies have investigated its administration by parenteral routes in admixture with various antigens. MPL has also been demonstrated to have a potent mucosal adjuvant activity, which is most probably associated with expression of TLR4 on mucosal tissues (144). Preclinical mucosal immunization studies have been performed by the nasal routes for hepatitis B, influenza and tetanus (145), HIV-1 (25), Streptococcus mutans (146), and by the oral route for Mycobacterium tuberculosis (147). In general, the results of administration by these mucosal routes was reported to result in the induction of antigen-specific mucosal IgA and systemic IgG antibodies as well as increased levels of cell-mediated immunity. Because of the unique biological effects, and strong adjuvant activity of MPL, investigations have sought to produce self-adjuvanting vaccines by conjugating various antigens to MPL (or MPL analogues). In one study a trinitrophenyl (TNP) group was attached via a 6-aminocaproic acid linker to Escherichia coli J5 MPL. Intraperitoneal immunization of mice with this construct yielded high titers of anti-TNP IgM and IgG antibodies (148), suggesting that MPL can act as a carrier as well as an adjuvant when conjugated to haptenic antigens.

CONCLUSION The feasibility of lipopeptide-based vaccines has been demonstrated by numerous groups, and we are now seeing some evidence of the proof of principle of the approach where totally synthetic epitope-based vaccines that carry a lipid moiety have entered clinical trials. Among the first human studies was the demonstration that hepatitis B virus-specific CTL responses could be induced by lipopeptide vaccination and that these were of comparable magnitude to those elicited by acute viral infection (149–151). In addition, the French National Agency for AIDS Research (ANRS) have been developing T-cell-inducing lipopeptide vaccines against HIV since 1994 (reviewed in Ref. 152). In a series of trials, over 200 healthy volunteers and 48 infected patients have been vaccinated with lipopeptides alone, in combination with adjuvants or in a prime-boost regimen with canary pox vectored antigen. A common theme from these studies is that of safety and immunogenicity of lipopeptide vaccines. Whether they will impact on the viral load in chronic disease is the next hurdle, and potency will have to be

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maximized (153). We (154,155) are currently testing a lipopeptide-based vaccine candidate for hepatitis C in humans using a strategy of maturing and antigen loading patients monocytederived DC in vitro with lipopeptides and reintroducing them back into the patient. As hepatitis C virus-infected DC are compromised in their ability to mature, this strategy, which targets in vitro generated DC will hopefully provide potent APC to boost the patient’s own inadequate CTL responses or prime new T cells. CTL-inducing lipopeptides may also show therapeutic utility for established cancers such as cervical carcinoma where target antigens are known (156). However, this will need to be done early while the cancer cells still maintain intact antigen-processing and presentation pathways. Peptides are the currency of conversation between APCs and cells of the adaptive immune system. It seems inevitable therefore that synthetic epitopes should be the basis of a class of vaccines. As our knowledge of the nature of DC receptors and their ligands (principally the TLR s and C type lectins) increases, we become more competent at targeting APCs. Totally synthetic vaccine approaches are particularly amenable to the approach and the results that have been obtained with them make it inevitable that lipopeptide-based vaccines will appear in the market in the near future.

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the TLR 2/6 agonist MALP-2 as mucosal adjuvant. Vaccine 2006; 24(25):5269–5276. Luhrmann A, Tschernig T, Pabst R, et al. Improved intranasal immunization with live-attenuated measles virus after coinoculation of the lipopeptide MALP-2. Vaccine 2005; 23(39): 4721–4726. Ulrich JT, Myers KR. Monophosphoryl lipid A as an adjuvant. Past experiences and new directions. Pharm Biotechnol 1995; 6:495–524. Persing DH, McGowan P, Evans JT, et al. Toll-like receptor 4 agonists as vaccine adjuvants. In: Virgil EJC, O’Hagan DT, eds. Immunopotentiators in modern vaccines. Burlington, MA: Elsevier Academic Press, 2006:93–108. Jiang ZH, Bach MV, Budzynski WA, et al. Lipid A structures containing novel lipid moieties: synthesis and adjuvant properties. Bioorg Med Chem Lett 2002; 12(16):2193–2196. Johnson DA, Sowell CG, Johnson CL, et al. Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4-phosphates (AGPs). Bioorg Med Chem Lett 1999; 9(15):2273–2278. Baldridge JR, McGowan P, Evans JT, et al. Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin Biol Therap 2004; 4(7): 1129–1138. Baldridge JR, Yorgensen Y, Ward JR, et al. Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. Vaccine 2000; 18(22): 2416–2425. Childers NK, Miller KL, Tong G, et al. Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen. Infect Immun 2000; 68 (10):5509–5516. Doherty TM, Olsen AW, van Pinxteren L, et al. Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis. Infect Immun 2002; 70(6):3111–3121. Myers KR, Beining P, Betts M, et al. Monophosphoryl lipid A behaves as a T-cell-independent type 1 carrier for hapten-specific antibody responses in mice. Infect Immun 1995; 63(1):168–174. Vitiello A, Ishioka G, Grey HM, et al. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans. J Clin Invest 1995; 95(1):341–349. Livingston BD, Crimi C, Grey H, et al. The hepatitis B virusspecific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection. J Immunol 1997; 159(3):1383–1392. Livingston BD, Crimi C, Fikes J, et al. Immunization with the HBV core 18-27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules. Hum Immunol 1999; 60(11):1013–1017. Durier C, Launay O, Meiffredy V, et al. Clinical safety of HIV lipopeptides used as vaccines in healthy volunteers and HIVinfected adults. Aids 2006; 20(7):1039–1049. Seth A, Yasutomi Y, Jacoby H, et al. Evaluation of a lipopeptide immunogen as a therapeutic in HIV type 1-seropositive individuals. AIDS Res Hum Retroviruses 2000; 16(4):337–343. Gowans EJ, Jones KL, Bharadwaj M, et al. Prospects for dendritic cell vaccination in persistent infection with hepatitis C virus. J Clin Virol 2004; 30(4):283–290. Jackson DC, Deliyannis G, Eriksson E, et al. Dendritic cell immunotherapy of hepatitis C virus infection: toxicology of lipopeptide-loaded dendritic cells. Int J Peptide Res Ther 2005; 11:1–12. Steller MA, Gurski KJ, Murakami M, et al. Cell-mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7. Clin Cancer Res 1998; 4(9):2103–2109.

32 Vaccines Based on Dendritic Cell Biology Silvia B. Boscardin and Michel C. Nussenzweig Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, U.S.A.

Christine Trumpfheller and Ralph M. Steinman Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York, U.S.A.

INTRODUCTION Vaccine development against many global infectious diseases as well as cancer will likely require strategies that lead to strong T cell immunity. Dendritic cells (DCs) are antigen-presenting cells that induce strong adaptive immunity and memory, particularly T cell–based responses, and are therefore an attractive target for studies of vaccine biology and the development of more effective vaccines. In humans, DC-based vaccine strategies have to date used DCs that are loaded ex vivo with vaccine antigens and then reinfused, primarily in the setting of advanced cancer. This strategy will be reviewed briefly here but was the subject of a chapter in a prior edition of this textbook (1). Here we will emphasize a new approach that is the subject of preclinical studies in mice, which is to directly target vaccine proteins to DCs in vivo. The latter approach has the advantage over the former of being an off-the-shelf product rather than a patientspecific treatment. The most successful vaccines to date are comprised of either attenuated or inactivated pathogens, for example, the Sabin and Salk polio vaccines, or recombinant or purified portions of a microbe, for example, the hepatitis B vaccine or the split influenza vaccine, respectively. Microbe-based vaccines may not be feasible or effective for several prevalent problems such as AIDS, malaria, tuberculosis and cancer. This chapter considers vaccines comprised of microbial proteins and designed on immunological principles based upon the biology of DCs. To understand the rationale, we will first outline some intrinsic features of DCs that are important for the control of immunity: (i) their location and movements in vivo, which allows DCs to act as sentinels for antigen capture and clonal selection of T cells; (ii) the repertoire of antigen receptors expressed by DCs, which allow for greatly improved uptake of vaccine proteins; and (iii) maturation in response to an array of immunologically relevant stimuli, which allow DCs to control the quality of the immune response.

INNATE FUNCTIONS OF DENDRITIC CELLS THAT LEAD TO THE CONTROL OF ADAPTIVE IMMUNITY Positioning and Homing of DCs DCs are positioned along body surfaces, often intimately associated with the epithelium, and they are able to home to the T-cell areas of lymphoid organs. This distribution and movement is an important feature of the DC lineage. It allows DCs to

sample environmental and self-proteins in the steady state, that is, in the absence of inflammation or infection, for the purpose of tolerance, while under conditions of perturbation, microbial and other antigens are presented for the purpose of immunity (2–4). DC migration into lymphoid tissues allows for productive interactions with T cells. This can now be visualized in living lymph nodes by intravital two-photon microscopy. Migrating mature DCs arrive in the T-cell area where they efficiently select T cells specific for the presented antigens (5–7). In the T-cell area, these DCs join a network that is already present in the steady state (8). Stable cell-cell contacts develop when antigen-bearing DCs encounter their cognate T cells, and these contacts persist at least 18 hours. Such contacts are apparent in the steady state, when DCs can be tolerogenic, and upon DC maturation, when immunity develops (9,10). In summary, the unique distribution of DCs positions them to capture antigens in peripheral tissues and then move to lymphoid organs. There, in the T-cell areas, DCs scan T cells circulating through lymphoid tissues and select antigen-specific clones from the repertoire, leading to the induction of either peripheral tolerance or immunity, as we will stress below.

DC Receptors and Their Expression by Different DC Subtypes DCs express a large number of endocytic receptors capable of mediating adsorptive uptake. Many of these are C-type lectins, which can either be type II transmembrane proteins with a single, carboxyl terminal lectin domain, for example, Langerin/ CD207, DC-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN)/CD209, BDCA-2, DC-associated C-type lectin-1 (Dectin-1), DC inhibitory receptor-2 (DCIR-2), or type I proteins with multiple lectin domains, for example, mannose receptor (MR)/CD206, DEC-205/CD205. Additional endocytic receptors are FcgRs, which mediate presentation of immune complexes and antibody-coated tumor cells on both major histocompatibility complex (MHC) class I and II. DCs also capture dying cells, although the precise receptors that are employed are a subject of current research. Interestingly, individual receptors can be expressed on distinct subsets of DCs. For example, Langerin/CD207 and DEC-205/CD205 are expressed on Langerhans cells (LCs) (11), while DC-SIGN/CD209 and MR/CD206 are highly expressed on dermal DCs (12) and monocyte-derived DCs (13). In mice, the CD8a-positive subset of DCs expresses

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DEC-205 as well as Langerin (14,15), while the CD8-negative subset is DCIR2 positive (16). The latter receptor was recently recognized to carry the antigen identified by the monoclonal antibody 33D1 (16), which was the first DC-restricted mAb to be identified (17,18). The presence of numerous uptake receptors enables DCs to efficiently take up many different ligands, but the receptors also mediate different outcomes of antigen presentation and processing. We will consider four examples. First, individual receptors can follow distinct trafficking paths dictated by cytosolic domain sequences. DEC-205/CD205 has a stretch of three acidic amino acids that allows the receptor to target and slowly recycle through MHC class II-positive late endosomes. In contrast, MR/CD206 recycles quickly through cells via early endosomes, as is the case for many other adsorptive endocytosis receptors. A second feature of receptor function of DCs is that endocytosed antigen, for example, via DEC-205 or FcgRs, can be cross-presented on MHC class I. Cross presentation allows nonreplicating antigens to be captured and presented to CD8þ T cells. In contrast to the classical pathway for presentation on MHC I, cross-presented peptides do not need to be synthesized in the DCs, but instead ‘‘cross’’ to the MHC I products of the DC from another source, for example, from selected proteins (19–21), tumor cells (22–24), inactivated virus or dying infected cells (25–27), immune complexes (28–30), and self-tissues (31). The CD8a-positive, DEC- 205-positive subset of DCs in mice is the most efficient cell type for cross presentation, and this is currently explained by the high expression of proteins involved in class I presentation such as TAP and tapasin (16). The precise pathway underlying cross presentation is not yet fully defined (32–34), although distinct intracellular compartments are thought to be required (35). Successful protein-based vaccines for T cell–mediated immunity will likely have to harness the cross presentation pathway in vivo. Third, distinct uptake receptors can be expressed on distinct subsets of DCs, which in turn may influence the subsequent processing and presentation of antigen. By using antigens fused to either anti-DEC or 33D1 antibodies, Dudziak et al. (16) showed that the CD8þ DEC205þ DC subset presents antigens to CD8þ T cells more efficiently than the CD8 33D1þ subset. On the other hand, the CD8 33D1þ subset excels in rapidly presenting antigens to CD4þ T cells. These characteristics seem to be due to the fact that the CD8 33D1þ DCs express higher levels of lysosomal proteases that process antigens for loading onto MHC II, while the DEC205þCD8þ DCs retain the intact antigen for longer periods of time and as mentioned, have higher levels of the machinery for MHC I presentation. DC subsets exhibit other distinct functions. An example has been described by Soares et al. (36), who delivered a Leishmania antigen, LACK, within anti-DEC-205 and 33D1 fusion antibodies. Targeting of antigen to either DC subset in vivo led to efficient presentation to CD4þ T cells, but the DEC205þ DC subset exclusively induced the helper cells to produce IFN-g in an interleukin (IL)-12-independent but CD70- dependent manner; in contrast, the 33D1þ subset also induced some IFN-g in vivo but required IL-12, not CD70, and the subset induced T cells producing IL-4. Human tissues need to be studied to see if they conserve these features of DC subset function. Fourth, uptake receptors can associate with other signaling molecules. For example, dectin-1 binds yeast and zymosan particles and associates with toll-like receptor (TLR)-2, thereby

signaling TNF-a and IL-12 production (37,38). Dectin-1 also has an immunoreceptor tyrosine–based activation (ITAM) motif that, following phosphorylation, attracts the src kinase, syk, and mediates production of two other cytokines, IL-1 and IL-10 (39). In summary, the expression of numerous receptors enables DCs to efficiently capture antigens and process these to peptides for presentation on both MHC class I and II products to CD8þ and CD4þ T cells, respectively. The type of receptor targeted might have a role in vaccine antigen presentation and could dictate the functional features of the immune response.

DC-Mediated Tolerance and Maturation DCs play a central role in the induction of antigen-specific tolerance in central lymphoid organs and in the periphery. In the thymic medulla, DCs generate tolerance by deleting selfreactive T cells (40,41) and can also generate suppressor T cells or Treg (42). In addition, DCs are involved in peripheral tolerance to avoid reactivity to either self-antigens that escape negative selection (43) or never reach the thymus, or harmless environmental proteins, to which reactivity must be avoided. In the steady state, the targeting of antigens to DC uptake receptors, even with low doses of antigens, can lead to deletion of corresponding T cells (44–46). DCs also can contribute to peripheral tolerance by promoting the expansion and differentiation of T cells that regulate or suppress other immune cells (47–51). Efficient peripheral tolerance mechanisms are especially important at sites of infection, where DCs simultaneously process and present both self- and nonself-antigens. In contrast, DCs can be induced to differentiate or mature to induce strong effector T cell responses. DCs undergo maturation in response to many stimuli, ranging from microbial ligands for TLRs to many nonmicrobial stimuli such as CD40 ligation, necrosis, innate lymphocytes, and immune complexes. Maturation results in several phenotypic changes that are linked to an enhanced ability to process antigens and activate T cells. These phenotypic changes include increased production of MHC-peptide complexes (52), increased expression of T-cell binding (e.g., CD48 and CD58), and co-stimulatory molecules (e.g., CD80, CD86, TNF family members, notch ligands, T-bet transcription factor) (53,54), and production of chemokines (55) as well as large amounts of immune enhancing cytokines like IL-12 (56) and type I interferons (IFNs) (57). Importantly, distinct maturation stimuli can influence the outcome of DC-T cell interactions. A good example would be the myeloid DCs in human blood. When these cells encounter two different stimuli, thymic stromal lymphopoietin (TSLP) or CD40L, the mature DCs look very similar. They have heightened MHC class II and B7 expression and are highly dendritic. However, TSLP DCs cause naı¨ve T cells to differentiate into inflammatory Th2 cells that produce TNF in addition to IL-4, 5, and 13, while CD40L DCs cause naı¨ve T cells to differentiate into Th1 cells (58). Deeper analysis reveals that TSLP DCs make distinct chemokines from CD40L DCs and fail to make inflammatory cytokines like IL-1, IL-6, and IL-12. DCs respond quickly to several ligands for TLRs, which are germline-encoded innate receptors for microbial products. Interestingly, DC subsets can vary in their expression of TLRs and therefore respond to different microbial stimuli. TLR 7 and TLR9, the TLRs that respond to nucleic acids, are primarily expressed on plasmacytoid DCs and mediate the production of large amounts of type I IFNs. TLR3 is expressed at high levels

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on a subset of myeloid DCs, the CD8aþ subset in mouse lymphoid tissues, for example. The latter, however, does not express TLR 5 and TLR7 (59). TLR signaling can lead to the production of cytokines with significant immune enhancing effects. For example, IL-12, whose production is enhanced by the transcription factor IRF-5 (60), acts on CD4þ T cells to enhance Th1 differentiation. Type I IFNs (many IFN-as and a single IFN-b), whose production is enhanced by the transcription factors IRF-3 and IRF-7 (61), act on CD8þ T cells (62,63) and B cells (64) to enhance the development of cytotoxic T lymphocytes (CTLs), antibody formation, and memory. The type of TLR ligand also influences the differentiation of helper T cells. CpG DNA, a TLR 9 ligand, and imiquimod, a TLR 7 ligand, can be adjuvants for Th1-type immune responses (65,66), whereas in contrast, the TLR 2 ligand, Pam3Cys, and the TLR 5 ligand, flagellin, have been reported to favor Th2 type responses (67–69). In summary, the outcome of antigen presentation by DCs depends on the state of DC differentiation or maturation. In the steady state, immature DCs capture, process, and present a variety of environmental antigens and dying cells. The presentation of MHC-antigen complexes by immature DCs to T cells leads to tolerance, whereas mature activated DCs typically induce strong effector T-cell responses. DCs undergo terminal differentiation or maturation in response to a variety of environmental stimuli. The maturation program varies with the stimulus, and the consequences for lymphocytes are likewise different. Therefore, there is a need to dissect the immune responses that are induced by the engagement of different maturation stimuli so that vaccines can be designed to elicit responses that are appropriate to the pathogen at hand.

VACCINES COMPRISED OF DCS EXPOSED TO ANTIGENS EX VIVO Mature DCs are Adjuvants for Immune Responses After it became apparent that DCs were specialized and potent stimulators of T-cell mediated immunity in tissue culture, it was decided to use these cells as adjuvants in vivo in rodents. Lechler and Batchelor (70) showed that DCs were major stimulators of graft rejection in vivo and at small doses. Macatonia et al. tested hapten-modified DCs in vivo, and the cells induced contact sensitivity (71). Inaba et al. pulsed DCs ex vivo with protein antigens, reinfused the cells, and found that mice could be primed directly and specifically to the protein antigen captured by the DCs. In the latter experiments, the proteins first had to be given to the DCs in their immature or antigencapturing state (which is the state of most DCs in vivo), and then the maturing DCs were injected (72). Investigators next considered more challenging antigenic targets. Again the DCs served as adjuvants for strong T-cell priming. DCs pulsed with tumor antigenic peptides or with viral vectors recombinant for tumor antigens were able to elicit protective immunity to tumor challenge, and in some instances caused existing tumors to undergo some regression (73,74). DCs pulsed with microbial antigens also could induce protective immunity to infection (75,76), while DCs bearing autoantigens could trigger autoimmunity (77,78). Because it is well known that proteins and preprocessed antigenic peptides are poor immunogens unless they are administered together with adjuvants, these early experiments implied that DCs could function as ‘‘nature’s adjuvants’’ for inducing protective and pathogenic T cell– mediated immunity in vivo (72).

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Ex Vivo-Derived, Antigen-Loaded DCs in Cancer Therapy Once it was established that DCs could prime or sensitize T cells, after being charged with model antigens like OVA or keyhole limpet hemocyanin (KLH) and reinfused into mice, researchers decided to move this strategy into humans. To do so, one major challenge was to obtain large numbers of DCs for purposes of therapeutic vaccination or immunotherapy in the setting of cancer. Granulocyte-macrophage colony stimulating factor (GM-CSF) is a valuable cytokine for DCs (79,80), being used to expand DCs from proliferating progenitors (81–83), and for differentiating DCs from nonproliferating monocyte precursors (84,85). Much of the current research is being carried out using monocyte-derived DCs (86–88), which are potent and homogenous stimulators of immunity. It is possible to obtain populations of immature DCs by exposing monocytes to GM-CSF and IL-4 (89,90), and these can be differentiated into mature DCs by various stimuli such as TLR ligands, inflammatory cytokines, or CD40L (85,91). Some of the first studies to assess if autologous DCs could act as immune adjuvants were carried out in healthy volunteers. DCs were pulsed with KLH or with an influenza virus peptide ex vivo and reinjected in the absence of any other adjuvant. The antigens by themselves were not immunogenic, but on DCs, T-cell immunity was induced. Interestingly, the CD4þ T-cell response to KLH was Th1 in nature (92), while the memory CD8þ T-cell response to influenza matrix peptide seemed to select for higher affinity T cells (93). On the other hand, when immature DCs were used, antigen specific IL-10 producing cells were generated and could suppress the response of IFN-g producing T cells (23,92). The first clinical trial using monocyte-derived DCs was performed by Nestle et al. in 1998 (94). These authors loaded DCs with melanoma antigens (tumor lysate) and reported that 30% of stage IV patients were able to mount a response. Thurner et al. reported that the loading of mature DCs with a melanoma-specific peptide (Mage 3A1) expanded specific cytotoxic T cells; this occasionally caused the regression of some metastases (95) and frequently primed for Th1 type CD4þ T-cell immunity (96). Besides monocyte-derived DCs, peripheral-blood DCs loaded with specific idiotype protein have been used as vaccines in patients with follicular B cell lymphoma (97). Also, DCs derived from CD34þ hematopoietic progenitor cells were loaded with melanoma antigens and used to vaccinate patients. The individuals who survived longest were the ones able to mount a response against more than two melanoma antigens (98). Many phase I type safety studies have used DCs in advanced cancer patients (for a review see Ref.99). However, a major obstacle resides in the fact that the injected DCs home poorly to lymphoid tissues, thus failing to harness one of the fundamental features of DC function.

TARGETING OF TUMOR CELL VACCINES TO DCS IN VIVO Vaccines Comprised of Irradiated Tumor Cells Transduced with Cytokines Dranoff et al. have asked if irradiated tumor cells could acquire increased immunogenicity if transduced to express cytokine genes. Among a variety of tested cytokines, irradiated tumor cells expressing murine GM-CSF stimulated potent, long lasting, and specific antitumor immunity, requiring both CD4þ and

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CD8þ cells (100). When tested in metastatic melanoma patients, autologous irradiated GM-CSF transduced tumor cells, known as GVAX, were immunogenic, and biopsies of the vaccine site indicated that patient DCs were being recruited and expressed high levels of B7.1 (101). Jaffee et al. used allogeneic GM-CSFtransduced tumor cell lines as a vaccine and found that antigens within the injected irradiated GVAX were being crosspresented, again suggesting that host DCs were capturing the dying tumor cells and initiating CD8þ T-cell immunity (102). GVAX approaches are now in phase III trials.

Vaccines Comprised of Dying Tumor Cells and A Stimulus for NKT Lymphocytes A critical step in vaccine efficacy is that DCs capturing vaccines must also undergo maturation to be able to induce T-cell immunity and control its quality. Evidence for DC maturation in vivo was obtained when mice were injected with lipopolysaccharide (LPS) (103) and CpG (104). Before LPS treatment, many splenic DCs were found at the margin between the red and white pulp and could process soluble proteins effectively. On the other hand, six hours after LPS administration, DCs were found in increased numbers in the T-cell area, had a reduced capacity to process proteins, but showed increased expression of B7 costimulators and T-cell stimulatory capacity. It was also shown that plasmid DNA containing CpG motifs and a transcription unit for OVA could mature plasmacytoid and myeloid DCs in vitro (105). Both DC subsets upregulated the expression of costimulatory molecules CD86 and CD40 and could produce IL-6. In vivo, however, even upon repeated vaccination with plasmid DNA, clonal expansion of OVAspecific CD8þ T cells was comparable in TLR9-positive mice to TLR9-negative or MyD88-negative animals. Fujii et al. explored the capacity of maturing DCs to present tumor cells using irradiated transplantable mouse tumor cell lines as a source of antigen and NKT lymphocytes as a maturation stimulus. The NKT cells were activated with the CD1d binding glycolipid, a-galactosyl ceramide. The maturing antigen capturing DCs in vivo were able to adoptively transfer immunity to naı¨ve mice, which did not need to receive an additional exposure to either antigen or a-galactosyl ceramide. Interestingly, the capacity of DCs to elicit immunity required a CD40-CD40L signal that acted after the presentation of antigen and the upregulation of CD86 costimulatory molecules (106). This means that the induction of T-cell immunity by DCs in vivo is not simply dependent on ‘‘signal one,’’ which is MHC peptide and ‘‘signal two,’’ which is costimulation via B7 molecules. In addition, CD40L can be required, and this can be delivered by CD40L expressing T cells including activated NKT cells. These findings have been extended to tumor cell vaccines in mice. Liu et al. injected irradiated tumor cells intravenously, together with a-galactosyl ceramide and induced strong and long-lasting combined CD4þ and CD8þ T-cell immunity (107). By isolating the DCs capturing tumor antigens, they were able to link the innate NKT response to strong adaptive immunity in vivo, and this did not require that the tumor cells be modified genetically to express cytokines or other immune enhancing molecules. A more recent discovery by Shimizu et al. is that tumor cells could themselves be loaded with a-galactosyl ceramide and injected intravenously. The tumor cells could activate NKT cells and, following killing, were captured by DCs, which cross-presented the glycolipid to recruit NKT cells that matured the tumor antigen–capturing DCs; this initiated particularly strong T-cell

immunity, even with a single low dose of irradiated tumor cells (108). These findings, which have only been tested in mice, suggest a path to the induction of tumor immunity in patients that harnesses the basic principles of DC biology that were outlined above. Intravenous injection of dying tumor cells leads to uptake by DCs, and these DCs can be matured to energize CD4þ and CD8þ adaptive T-cell immunity.

DIRECT TARGETING OF VACCINE PROTEINS TO MATURING DCS IN VIVO: THE DEC-205/CD205 EXAMPLE A new approach to develop vaccine science and improve efficacy involves targeting of vaccine antigens to uptake receptors that are expressed by DCs in lymphoid organs. One method involves the use of antibodies to DC receptors, in which the antibodies are coupled or engineered to include vaccine proteins (Fig. 1). Antibodies target quickly and selectively to large numbers of DCs in lymphoid tissues, thus bypassing the need for DCs in the periphery to capture the vaccine and migrate to the T-cell areas. Several receptors have begun to be investigated and illustrate that receptor-based targeting of vaccines allows for greatly improved antigen presentation in vivo, by 100-fold or more relative to nontargeted vaccine protein.

Tolerance and Immunity Outcomes After Antigen Targeting to DEC-205/CD205 Hawiger et al. (44) and Bonifaz et al. (46) selectively delivered model antigens to DCs in vivo via the DEC-205/CD205 adsorptive endocytosis receptor using an anti-DEC antibody coupled to the antigen. As mentioned previously, the DEC-205 receptor is present in the CD8a-positive murine DC subset (14). In humans, this receptor is found in monocyte-derived DCs (109) as well as DCs in the T-cell areas of lymphoid tissues like lymph node (110) and spleen (111). Two different strategies were used. Hawiger et al. (44) cloned the heavy chain of the anti-DEC-205 antibody sequence and introduced in frame sequences for a hen egg lysozyme (HEL) peptide. The variable regions of both heavy and light chains of the anti-DEC-205 antibody (NLDC-145) were cloned in frame with mouse Ig kappa constant regions and IgG1 constant regions, the latter carrying mutations to reduce Fc receptors (FcR) binding (112). The antibody was produced by transient transfection of 293 cells and purified using protein G columns. When the antibody anti-DEC-HEL was administered to mice, the authors showed that the T cells were induced to expand but not polarized to produce the T helper type 1 cytokine IFN-g, and the activation response was not sustained. Within seven days, the number of antigen-specific T cells was severely reduced, and the residual T cells became unresponsive or tolerant to systemic challenge with antigen in complete Freund’s adjuvant (CFA). On the other hand, the co-injection of the DC-targeted antigen and anti-CD40 agonistic antibody changed the outcome from tolerance to prolonged T-cell activation and immunity. Therefore, the authors concluded that in the absence of additional stimuli, DCs induce transient antigen-specific T-cell activation followed by T-cell deletion and unresponsiveness (Table 1). Interestingly, deletion was not the only mechanism by which T cells could be tolerized by targeting antigens directly to DCs under steady state. In another study (113), the targeting of the myelin oligodendrocyte glycoprotein (MOG) peptide

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Figure 1 Production of the recombinant anti-DEC antibody fused to the antigen of interest. (A) Schematic representation of the steps involved in the fusion antibody production. 293T cells are co-transfected with plasmids encoding the light and heavy chains of the anti-DEC205 antibody. Fusion antibodies are subsequently purified by protein G purification. (B) Antibody integrity is assessed by SDS-PAGE followed by Coomassie staining.

directly to DCs resulted in a novel form of peripheral T-cell tolerance that was sufficiently profound to prevent autoimmune experimental acute encephalomyelitis. The tolerized T cells did not respond to challenge with the antigen in vivo but were not intrinsically anergic because they remained highly responsive to T-cell receptor stimulation in vitro. The mechanism involved in this type of tolerance requires that DCs induce CD5 expression on activated T cells. Bonifaz et al. (46) chemically coupled anti-DEC-205 antibody to the ovalbumin (OVA) protein. The OVA delivered to DCs in vivo was efficiently processed and presented to both CD4þ and CD8þ T cells. When DCs were targeted with antigen in the steady state, they induced peripheral tolerance. In contrast, combined administration of DC-targeted antigen with a DC maturation stimulus, agonistic anti-CD40 antibody, led to prolonged CD4þ and CD8þ T-cell activation (44,46). Furthermore, the immunity induced by DEC targeting was longer lasting and more effective than administration of classical adjuvants such as CFA. As shown for the T cells, the administration of anti-DEC-OVA in the absence of a maturation stimulus was unable to induce antibodies against the OVA protein (Fig. 2). Subsequent studies have emphasized the introduction of antigens into antibodies by genetic engineering of the heavy chain rather than chemical conjugation, because the former method

reliably and stoichimetrically introduces one molecule of vaccine antigen into each heavy chain molecule.

Anti-DEC-205 Hybrid Antibodies as Vaccines The above results opened the possibility of using this DC antibody targeting strategy for vaccination protocols in mice with a native polyclonal repertoire rather than adoptively transferred TCR transgenic T cells. Bonifaz et al. (114) demonstrated that one could improve T-cell vaccination using antibodies directed to DEC-205. Mice immunized with the same anti-DEC-205 antibody conjugated to OVA in the presence of the maturation stimulus anti-CD40 developed strong T-cell immunity and resisted OVA-modified tumor cells as well as a challenge of recombinant vaccinia-OVA virus. To determine whether antigens delivered to DCs in lymphoid organs induced T-cell help for antibody responses, Boscardin et al. (115) used a hybrid anti-DEC-OVA antibody that was administered in the presence of anti-CD40 plus poly I: C as DC maturation stimuli. These authors targeted OVA to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, 4-hydroxy-3-nitrophenyl acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T-cell helper responses to the carrier

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Table 1 Antibodies Used for Targeting Antigens Directly to DCs Receptor

Cell type expressing the receptor

Monoclonal antibody used for targeting

DEC-205/CD205

DC

NLDC-145

MMR/CD206

DC Macrophages Lymphatic/hepatic endothelia

DC-SIGN/CD209

DC DC-like cells DC Macrophages/monocytes Neutrophils DC Macrophages DCs Monocytes/macrophages DCs DCs

Dectin 1

Dectin 2 FIRE CIRE Fc receptors LOX-1

DCs Macrophages/monocytes Endothelial cells Fibroblasts

Protein/peptide targeted

Reference

Hen egg lysozyme Myelin oligodendrocyte glycoprotein OVA HIV GAG Plasmodium Circumsporozoite Protein Leishmania LACK HIV GAG Melanoma pmel17 OVA

36 118 124 125

3.29 AZN-D1 E10 2A11

HIV GAG Keyhole limpet hemocyanin TT peptide OVA

118 136 137 139

11E4

OVA

140

6F12

Rat monoclonal antibodies

143

5H10 D-DC8.3

Rat monoclonal antibodies TT peptide Hepatitis C virus peptide OVA

143 145 145 148

MG38.2 B11

23C11

44 113 114 116 115

This table shows some antibodies used to date to target DCs either in mice or in humans. Abbreviations: DCs, dendritic cells; OVA, ovalbumin, TT, tetanus toxoid; FIRE, F4/80-like-receptor; CIRE, C-type lectin receptor.

Figure 2 Anti-DEC-OVA targeting to DCs induces antibody responses after boost with OVA protein. C57BL/6 mice were immunized with either 3 micrograms of anti-DEC-OVA antibody or control (Iso)-OVA in the presence or absence of 50 micrograms anti-CD40 and 50 micrograms of poly I:C. Eight weeks after the primary immunization, the mice were boosted with either 1 microgram of OVA-NP11 or PBS. Antibody titers against OVA, two weeks after the boost are shown. Immunization or priming conditions are shown in the legend. Symbols represent individual mice and the horizontal bars represent the mean value for each group. Abbreviations: DCs, dendritic cells; OVA, ovalbumin.

protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity anti-hapten immunoglobulins G (IgGs). Small doses of DC-targeted OVA induced high titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA adjuvanted with alum. Similar results were obtained when the circumsporozoite protein from Plasmodium yoelii was delivered to the DCs. The results led the authors to conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T-cell help for humoral immune responses. To assess the outcome of targeted delivery of HIV antigens, Trumpfheller et al. (116) delivered the HIV gag protein to DCs using the anti-DEC-205 antibody along with the maturation stimulus anti-CD40 and the TLR3 ligand poly I:C. The CD4þ T-cell immune response to DC-targeted gag was compared to other vaccine strategies. A single dose of DEC-205targeted HIV gag p24 or p41 induced stronger CD4þ T-cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of IFN-g and IL 2 producing CD4þ T cells were elicited, including double cytokine-producing cells. In addition, the response was broad because the primed mice responded to at least two peptides each in three different MHC haplotypes. Long-lived T-cell memory was also observed, and after subcutaneous vaccination, protection (which was CD4 and IFN-g dependent) developed to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. Interestingly, the authors showed in a follow-up study that poly I:C alone could serve as an adjuvant to allow a

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Figure 3 The quality of the HIV gag-specific CD4þ T cell response after prime-boost immunization with DEC-targeted vaccine and poly I:C. Balb/c mice were immunized with 50 mg polyI:C and 5 mg DEC-HIV gag p24 antibody IP, and boosted with the same immunization at 6 weeks after primary immunization. HIV gag-p24 specific cytokine production from splenocytes of vaccinated mice was determined at 2, 4, and 5 weeks after boost. Multiparametric flow cytometry was used to determine (A) the frequency of cells expressing each of the seven possible combinations of IFN-g, IL-2 and TNF-a and (B) the fraction of the total response comprising cells expressing all three cytokines (3þ), any two cytokines (2þ), or any one cytokine (1þ).

DC-targeted HIV gag p24 protein to induce protective Th1 CD4þ T cells (117). Following a prime-boost immunization with DEC-targeted HIV gag p24 along with poly I:C, the responder CD4þ T cells had qualitative features that have been correlated with protective function. This included the ability of the induced T cells to produce multiple cytokines (Fig. 3) and in high amounts for prolonged periods as well as their ability to proliferate and continue to secrete IFN-g in response to HIV gag p24. To begin to extend these ideas to humans, Bozzacco et al. used the DEC-205 receptor to deliver antigens to monocytederived DCs and then assessed the capacity of DEC-205 to mediate cross presentation to CD8þ T cells from HIV infected individuals (all of whom had CD4þ T-cell counts of >600/mL). Importantly, it was established that numerous different peptides from the HIV protein, gag p24 could be cross-presented by DCs expressing many allelic forms of human MHC I. These were the first data to show that cross presentation, here via DEC-205, mediated CD8þ T-cell immunity across a diverse spectrum of MHC haplotypes (118).

ANTIBODY TARGETING TO DIFFERENT DC SUBSETS: A COMPARISON OF ANTI-DEC-205 WITH 33D1 ANTIBODY TARGETING The first DC-specific mAb was termed 33D1 (17), and it was shown that 33D1 and DEC-205 (formerly NLDC-145) were expressed on different subsets of DCs in mouse spleen (18),

also termed CD8-negative and CD8-positive, respectively (119). Recently, Dudziak et al. cloned the antigen recognized by 33D1 mAb and found it to be a lectin, DCIR-2 (16). When they targeted OVA protein within 33D1 versus DEC-205 mAbs, they found that 33D1 was about 10-fold superior in stimulating the growth of CD4þ TCR transgenic T cells, while DEC-205 was about 10-fold superior in stimulation CD8þ TCR transgenic T cells (16). Part of the underlying mechanism is that 33D1 is expressed on a subset of DCs that is rich in the machinery for presentation on MHC II, while DEC-205 is expressed on DCs that are rich in mRNAs and proteins for MHC I processing. Soares et al. compared 33D1 and DEC-205 as targets for antibody-based delivery of LACK, an antigenic protein from Leishmania major. They confirmed that 33D1 was more efficient than DEC-205 in inducing the clonal expansion of LACKspecific transgenic, CD4þ T cells. However, when the CD4þ T-cell immune response that developed from a polyclonal repertoire was studied, several distinct features were noted in DC subset function. In Balb/c mice, which are prone to differentiate CD4þ T cells along a Th2 pathway, anti-DECLACK only induced Th1 immunity whereas 33D1-LACK elicited less IFN-g but also induced IL-4. Even more surprising was that the mechanism for Th1 immunity via anti-DEC-LACK did not require IL-12 but instead was fully dependent upon expression of CD70, a TNF family costimulator (36). In contrast, IFN-g production via 33D1-LACK was totally dependent upon the classical Th1 differentiation factor, IL-12. These data indicate that the receptor and DC subset that are targeted are

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both important variables that influence the outcome of selected delivery of vaccine proteins to DCs, and at the same time, one can probe DC function and mechanisms of action in intact tissues with the targeting approach.

OTHER POTENTIAL RECEPTORS FOR TARGETING PROTEIN VACCINES TO DENDRITIC CELLS Mannose Receptor/CD206 The MR (CD206) in the steady state is expressed at high levels on certain macrophages and lymphatic and hepatic endothelia (120). Different subsets of DCs also express the MR, primarily interstitial DCs (121) and CD8aþ splenic DCs (122). The MR recognizes carbohydrates like mannose, fructose, glucose, Nacetylglucosamine, and maltose present on the surfaces of bacteria and yeast (120). The function of the MR may explain why mannosylated peptides and proteins are able to stimulate MHC II restricted peptide specific T cells more efficiently than peptides and proteins that are not mannosylated (123). To specifically target the MR, Ramakrishna et al. (124) generated a monoclonal antibody specific for the human MR (clone B11) and genetically introduced a melanoma antigen, pmel17. Treatment of human DCs with the hybrid antibody resulted in the presentation of pmel17 in the context of HLA class I and class II molecules. Also, the CTLs generated were able to lyse HLA-matched targets. In one initial study to compare the outcome of targeting the MR to DEC-205 in vitro, the MR was less effective in inducing gag-specific CD8þ T-cell responses (118). In vivo targeting to the MR was also shown using a transgenic mouse model expressing the human MR (hMR Tg mice) (125). The administration of an anti-hMR hybrid antibody fused to OVA induced cellular immunity. The concomitant administration of the anti-hMR-OVA antibody with CpG was able to induce OVA-specific tumor immunity only in hMR Tg mice, while wild type mice remained unprotected. Importantly, McKenzie et al. (126) could increase the numbers of MR expressing DCs by administering LPS intravenously. When the antibodies were injected 10 minutes later, an increase in the targeting was observed. This illustrates that an adjuvant like LPS might alter the types of DCs that are available to induce vaccine immunity.

Langerin/CD207 Langerin is the lectin that mediates the formation of Birbeck granules, which are the hallmark structures of epidermal LCs (11). Langerin is now known to be expressed on DCs outside the epidermis, particularly the DEC205þ subset of DCs in spleen and lymph nodes (15,127,128), and more recently recognized dermal Langerinþ cells (129–131). A monoclonal antibody has been retrieved, which recognizes the external region of mouse Langerin (15). The heavy and light chains of this L31 mAb have been cloned, and OVA introduced into the heavy chain. Using these anti-CD207-OVA conjugates, Idoyaga et al. have found that Langerin mediates antigen presentation in mice on both MHC I and II products (132).

DC-SIGN/CD209 DC-SIGN/CD209 is expressed in large amounts on monocytederived DCs, but only on small numbers of DCs in the T-cell area of lymphoid tissues in the steady state (110,111). In skin

sections, DC-SIGN is only expressed on dermal DC, whereas CD1a-positive LCs in the epidermis are negative. Furthermore, DC-SIGN is expressed on DC-like cells present in the mucosal tissues, such as rectum, cervix, and uterus (133,134) as well as lung (135). The efficiency of targeting antigens to human DCs via DC-SIGN was evaluated using humanized anti-DC-SIGN antibody (hD1) chemically cross-linked with KLH (136). This chimeric antibody-protein complex (hD1-KLH) bound to DC-SIGN and was rapidly internalized. The DCs induced proliferation of patient PBMCs at 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1-KLH-targeted DCs induced proliferation of naı¨ve T cells recognizing KLH epitopes in the context of MHC I and II. In another study, Dakappagari et al. (137) used an antibody that cross-reacted with L-SIGN and DC-SIGN fused to a T helper epitope from tetanus toxoid (TT). A T-cell response was induced when the fusion antibody was targeted to DCs.

DECTIN-1 and -2 Both dectin-1 and 2 are expressed on DCs (dermal DCs and CD8a DCs), macrophages, neutrophils, and monocytes and are receptors for b-glucan-recognizing b1,3- and b1,6-linked glycans on yeast cell walls (138). The ability of dectin-1 and 2 to present antigen was studied using OVA conjugated to an antidectin antibody (139,140). Using adoptive transfer of transgenic OT-I T cells, these authors could show that a low dose of antidectin-OVA antibody (1 mg) was able to induce some expansion of OT-I T cells when compared with the protein alone. Also, only the conjugated antigen generated antigen-specific IFN-g producing cells.

FIRE and CIRE The F4/80-like-receptor (FIRE) is expressed specifically on CD8CD4þ and CD8CD4 DCs and weakly on monocytes and macrophages (141). C-type lectin receptor (CIRE), on the other hand is expressed by the same DC-subtypes as FIRE but not expressed by monocytes or macrophages (142). When antiFIRE and anti-CIRE rat monoclonal antibodies were used to immunize mice, anti-rat IgG titers were 100-fold greater than those obtained using nontargeted antibodies (143).

Fc Receptors FcRs bind immune complexes and mediate both effector and immune activating processes. There is one type of FcR for each class of immunoglobulin: FcaR (IgA), FceR (IgE), FcgR (IgG), and FcamR (IgA/IgM). In mice, there are four additional types of FcgRs: FcgRI, FcgRII, FcgRIII, and FcgRIV (144). Antigen presentation is facilitated by immune complexes via FcgR. In humans, the M-DC8þ DC subset present in peripheral blood mononuclear cells (PBMCs) expresses high levels of FcgRIII. When this receptor was targeted by an antibody anti-CD16 (FcgRIII specific) conjugated with a tetanus peptide or a hepatitis C virus peptide, the efficiency of activation of CD4þ T cells was 500 times superior when compared to the free antigen (145).

LOX-1 Scavenger receptors are cell-surface glycoproteins that bind modified lipoproteins and a broad spectrum of structurally unrelated ligands such as modified LDL (Ox- and Ac-LDL) (146), apoptotic cells, and bacteria-derived cell wall components like LPS and lipotechoic acid (147). LOX-1 is a scavenger

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receptor and is the main heat shock protein-binding structure on human DCs. Delneste et al. (148) used an anti-LOX-1 antibody coupled to OVA protein and showed that the administration of three doses of this mAb in the presence of an adjuvant could control the growth of an OVA bearing tumor in mice. Furthermore, the protection was mediated by CD8þ T cells because their ablation abolished the protective effect of the anti-LOX-1-OVA mAb. These results clearly showed that targeting of antigens to the LOX-1 scavenger receptor present at the surface of the DCs could induce cross presentation (148).

TARGETING OF DNA VACCINES TO DC RECEPTORS In principle, ligands and antibodies that bind to DC receptors can be used to increase the efficiency of other types of vaccines. A recent example relates to DNA vaccines. Nchinda et al. introduced the sequences for a single chain anti-DEC-205 antibody into DNA plasmids that were used to deliver either OVA or HIV gag p41 (149). They found that the single chain anti-DEC greatly improved the delivery of antigen to DCs in vivo as well as antigen presentation to CD4þ and CD8þ T cells, relative to single chain control Ig DNA vaccine. At the same time, they were able to reduce by 100-fold the amount of DNA that was required to induce T-cell immunity as well as protection to a challenge with vaccinia gag. Typically, DNA vaccines suffer from a need to use high doses of DNA and from a relatively weak T cell–mediated immune response. DC targeting may rescue these deficiencies.

CHALLENGES FOR THE DEVELOPMENT OF DC-TARGETED VACCINES The receptor-mediated delivery of vaccine proteins to DCs in situ enhances the quantity and quality of T-cell immunity that develops with protein and DNA-based vaccines. Improved antibody responses can also develop through the function of helper T cells. These improvements result from several features of targeted delivery. The receptors enhance the uptake of antigen by individual DCs. Also antibody delivers antigens rapidly and systemically to large numbers of DCs in the T cell areas of lymphoid organs, whereas relatively small numbers of DCs tend to pick up and transport particulate vaccines injected into the skin. However, receptor-mediated delivery of vaccine proteins to DCs in situ is still in its infancy, and several topics need to be unraveled in the future. First, immunological outcomes need to be compared following targeting of different receptors and DC subtypes, which as shown by Soares et al., can strongly bias the immune response to a Th1 type of immunity (36). Second, DCs need to be matured during vaccination in a way that is appropriate to the pathogen at hand. For many maturation stimuli, particularly for TLR ligands, more research is needed to assess efficacy in vivo as well as the specific changes in DCs required to generate protective immunity and memory. Third, there are still substantial gaps in understanding DC migration and traffic at mucosal surfaces. More research is needed to learn how DC function can be controlled to induce stronger mucosal immunity, which is a requirement for vaccines against many prevalent infections. Fourth, vaccine studies need to be accompanied by in-depth immune monitoring to define assays for protective lymphocytes. For example, in HIV vaccines, it is thought that protective T cells will produce several and higher

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levels of cytokines like IFN-g, IL-2, and TNF-a. Interestingly, as mentioned above, a prime-boost immunization with DEC205 targeted HIV gag p24 protein and the TLR3 ligand poly I:C as maturation stimulus in mice induces mainly multifunctional CD4þ T cells and T cells with proliferative capacity (Fig. 3) (117). Fifth, more research needs to be done with patients. The patient sets the standards for the quality of knowledge that is required to understand many aspects of disease and its treatment, for example, often the pathogen (tumor, microbe) is not easily or completely modeled in mice. Nevertheless, this review has considered preclinical information, which supports the promise of exploiting DC biology to design better vaccines.

SUMMARY We have considered preclinical information that supports the promise of exploiting DC biology to design better vaccines. One overall feature of DCs is that they are specialized to overcome the demands of initiating an adaptive immune response. Antigens including vaccines are typically deposited in peripheral tissue, and they need to gain access to lymphoid organs, where they must be recognized by rare clones of antigen-specific lymphocytes that continually circulate through these tissues. DCs provide a means of solving this challenge. They are sentinels in many respects. DCs capture, process, and present antigens so that they are displayed to the antigen receptors on T cells and elicit responses to a breadth or spectrum of antigenic peptides. DCs migrate to the T-cell areas of lymphoid organs, in a position to select rare, antigen-specific T cells. A second overall feature of DCs is that they control the quality of the immune response. In addition to specific antigens, DCs mature in response to stimuli from the microbes or other inflammatory insult, and this guides the appropriate adaptive T-cell response. These intrinsic features of DCs allow them to function as sentinels to initiate immunity to antigens in vaccines. Several strategies are being developed to harness DC biology in vaccine design. Of particular interest are attempts to develop protein and DNA-based vaccines that target vaccine antigens directly to maturing DCs in situ. One strategy is to incorporate vaccine proteins into monoclonal antibodies to antigen uptake receptors. The receptor-mediated delivery of vaccine proteins to DCs in situ greatly enhances vaccine efficacy in quantity and quality. These improvements result from several features of targeted delivery. First, the receptors enhance the uptake of antigen by individual DCs. Second, antibody delivers antigens rapidly and systemically to large numbers of DCs in the T cell areas of lymphoid organs, whereas relatively small numbers of DCs tend to pick up and transport particulate vaccines injected into the skin. Third, the subsets of DCs that have been targeted are capable of polarizing T cells along valuable lines, yielding Th1 type immunity along with memory. While this field is a new one, it is possible that DC targeted protein-based vaccines will prove to be effective, in contrast to the poor efficacy of nontargeted protein vaccines in the past. This would be valuable because in contrast to microbial vectors, protein-based vaccines should not elicit anti-vector immunity and could be used in several different clinical settings. In addition to the goal of improving the efficacy of vaccines, the targeting of antigens to DCs in vivo allows one to study DC biology in situ and identify principles that will likely contribute to vaccine design.

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33 Vaccinia Virus and Other Poxviruses as Live Vectors Bernard Moss Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

VACCINIA VIRUS: THE SMALLPOX VACCINE On May 14, 1796, Edward Jenner inoculated eight-year old James Phipps with cowpox virus, obtained from an infection on the hand of Sarah Nelmes, a milkmaid. This simple procedure was shown to provide complete protection against smallpox, and formed the basis for its ultimate eradication (1). The prophylactic effect of vaccination was due to the close genetic and antigenic relationships between variola virus, the causative agent of smallpox, and its more benign relatives, cowpox virus and vaccinia virus. The latter virus may have been isolated from an infected horse and, presumably because of its milder reactivity, was substituted for cowpox virus (2). Vaccinia virus was economical to produce, active in low amounts, heat stable, resistant to freeze drying, simple to administer, relatively safe, and provided long-lasting immunity. Of equal importance for smallpox eradication, however, were the ease of diagnosis of the disease, lack of antigenic variation, and the absence of latently infected human or animal reservoirs. Although the vaccine was immediately successful, eradication of smallpox proved difficult for logistical reasons. In 1967, the World Health Organization implemented a new intensified global ring vaccination strategy that ultimately contained and eliminated variola virus from nature. The last endemic case of smallpox occurred in 1977. Nevertheless, registered stocks of variola virus are preserved in both the United States and Russia, and there is a possibility of unregistered stocks elsewhere. With the eradication of smallpox, the need for vaccination was eliminated, and the practice largely stopped. Therefore, most people are now susceptible to variola virus, as well as other orthopoxviruses, such as monkeypox virus. However, as a precaution against the reintroduction of variola virus from an unregistered stock, a new tissue culture–derived vaccinia virus vaccine ACAM2000 has been developed and licensed in the United States (3). In addition, moderately and highly attenuated strains of vaccinia virus including LC16M8 (4) and modified vaccinia virus Ankara (MVA) (5) are being evaluated as safer alternatives to the conventional vaccine. Soon after the eradication of smallpox and the cessation of general vaccination, the ability to produce recombinant vaccinia viruses that express genes of other microorganisms was developed (6,7). Such genetically engineered viruses have been employed extensively as research tools to establish the targets of humoral and cell-mediated immunity and are being evaluated as live recombinant vaccines. Similar approaches were used to generate immunogenic avipoxvirus (8,9), capripoxvirus (10), and members of other poxvirus genera.

CONSTRUCTION OF POXVIRUS EXPRESSION VECTORS Insertion of Foreign DNA into the Poxvirus Genome The development of expression vectors depended on an understanding of the molecular biology of poxviruses, a subject which is reviewed in detail elsewhere (11). The distinctive characteristics of members of the poxvirus family include: a large complex enveloped virion containing enzymes for mRNA synthesis; a genome composed of a linear double-stranded DNA molecule of about 200,000 base pairs; and the ability to replicate within the cytoplasm of infected cells. Detailed protocols for preparing and characterizing recombinant vaccinia viruses are available (12–15), and only general concepts are dealt with here. The large size of the vaccinia virus genome posed an initial hurdle to the incorporation of foreign genetic material. In addition, the viral DNA is not infectious because enzymes contained within the virion are essential for gene expression. However, it was known that recombination occurs between homologous DNA sequences of poxviruses (16). Furthermore, recombination was shown to occur between virus-derived genomic DNA and either subgenomic DNA fragments (17,18) or recombinant plasmids (19) that had been transfected into the cell. The latter finding provided a way of inserting foreign DNA into the vaccinia virus genome: a plasmid containing a foreign gene flanked with vaccinia DNA is transfected into an infected cell allowing homologous recombination to occur during viral DNA replication. Of course, for the recombinant vaccinia virus to remain infectious, the foreign DNA must not interrupt any vital viral function. This was not an obstacle, however, because there are many nonessential vaccinia virus genes, and it is also possible to insert DNA between genes. In this manner, foreign DNA segments as large as 25,000 base pairs were recombined into the vaccinia virus genome (20). A variety of methods are available to select recombinant viruses or distinguish the plaques from the parental virus. Similar methods have been used to generate recombinant avian and other poxviruses. There are additional but less commonly used methods of forming recombinant vaccinia viruses. Two of these methods depend on the in vitro cleavage of the vaccinia virus genome at a unique restriction endonuclease site. In one procedure, a DNA fragment containing the foreign DNA is ligated to the cleaved segments, and then the full-length genome is transfected into cells infected with a helper virus—either a

Chapter 33: Vaccinia Virus and Other Poxviruses as Live Vectors

temperature-sensitive vaccinia virus or an avian poxvirus (21,22). The procedure is especially useful for very large DNA inserts, or to avoid intermediate cloning in bacteria. Alternatively, three-way recombination can be achieved by transfecting the cleaved genome and a foreign DNA segment with flanking vaccinia virus sequences into cells infected with a helper virus (23,24). The third method involves the insertion of genes into a circular bacterial artificial chromosome containing the entire vaccinia virus genome in Escherichia coli, which can then be rescued by transfection into mammalian cells with a helper poxvirus (25–27).

Expression of Foreign Genes Poxviruses encode their own transcription system, which includes a multisubunit RNA polymerase, stage-specific transcription factors, poly(A) polymerase, and capping and methylating enzymes (28). Because the DNA sequences that are recognized by the viral transcription system are unique, the use of poxvirus promoters for foreign gene expression is obligatory. Vaccinia virus gene expression is temporally regulated: early stage genes are transcribed before DNA replication, and intermediate and late stage genes are transcribed after DNA replication. Some genes are expressed throughout infection because they have two promoters. A foreign gene inserted into the vaccinia virus genome will be regulated in a predictable manner, depending on the vaccinia virus promoter placed adjacent to it. In general, high expression is obtained by using late promoters derived from vaccinia virus genes encoding major structural proteins, but early or early/late promoters are best for expression in antigen-presenting cells and inducing cytotoxic T-cell responses (29,30). For optimal results, synthetic early/late promoters have been designed (31,32). Since vaccinia virus can accommodate large amounts of additional DNA, multiple genes can be expressed using vaccinia virus vectors. Care needs to be taken in choosing the form of the foreign gene to be expressed. Most importantly, only continuous open reading frames may be used, as splicing does not occur in the cytoplasm. This problem is avoided by using cDNA copies of mRNAs. In addition, cryptic poxvirus early transcription termination signals, TTTTTNT in which N is any nucleotide, should be eliminated by silent mutation (33).

General Methods of Isolating Recombinant Poxviruses Vaccinia virus is cytopathic and produces large plaques on monolayers of appropriate cell lines; most virus isolation procedures depend on the discrimination of plaques formed by parental and recombinant viruses. Recombinant viruses can be recognized by the presence of foreign DNA sequences or expression of the gene products. Thus, plaques can be screened by hybridization to specific DNA (34) or by binding to antibody (12). However, because recombinants usually comprise less than 0.1% of the progeny, more convenient selection or screening methods have been devised and are described below. To facilitate the process of recombinant virus formation, plasmid transfer vectors have been constructed in which a vaccinia virus promoter followed by unique restriction endonuclease sites is flanked by vaccinia virus DNA needed for homologous recombination. The first of these general transfer vectors used the vaccinia virus thymidine kinase (TK) gene as the site for promoter insertion to provide a method for the

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selection of recombinant viruses (35). The selection depends on the disruption of the TK gene upon recombination of the foreign DNA into the vaccinia virus genome. The basis for the selection is the lethal effect of the incorporation of certain nucleoside analogs into viral DNA and the need for a functional TK for this to occur. Other selection approaches depend on the cotransfer of a dominant selectable marker along with the foreign gene. Genes encoding the neomycin-resistance (36), guanine phosphoribosyltransferase (37,38), hygromycin-resistance (39), puromycin-resistance (40), herpes simplex virus (HSV) TK (41), and TK/thymidylate kinase fusion gene (42) have been employed for this purpose. Several screening methods have been developed, some of which may be used in conjunction with selection procedures or as an alternative. These include the cotransfer of the b-galactosidase (43), b-glucuronidase (44), or enhanced green fluorescent protein (45) genes, which allow the color staining or fluorescence of recombinant plaques. Still other screening methods depend on the plaque size of the recombinant virus (46) or host range (47). For vaccine purposes, the marker genes can be removed by a recombination step (48). A convenient approach is to first insert the green fluorescent protein gene regulated by a vaccinia virus promoter, and then replace it with the gene of interest; the desired recombinant virus would make nonfluorescent plaques.

Hybrid Expression Vectors A high-expression system with great utility for protein synthesis in cultured cells, rather than for live virus immunization, has been developed. This innovation took advantage of the bacteriophage T7 (or related SP6) RNA polymerase, which is a single subunit enzyme with high catalytic activity and strict promoter specificity. By attaching a vaccinia virus promoter to the T7 or SP6 RNA polymerase gene, recombinant vaccinia viruses that express bacteriophage RNA polymerase have been constructed (49,50). Cells are infected with the latter and then either transfected with a plasmid that has a foreign gene regulated by the bacteriophage promoter or coinfected with a second vaccinia virus that has the bacteriophage promoter regulated foreign gene (49,51). Inducible systems have been developed in which both the T7 RNA polymerase gene and the T7 promoter regulated foreign gene are in the same virus (52). Expression can be enhanced by inserting the untranslated leader sequence of encephalomyocarditis virus before the initiation codon (53).

Fidelity of Expression Excellent results have been obtained using vaccinia virus vectors to express foreign genes from viral, prokaryotic, or eukaryotic sources. Factors that contribute to the high success rate include the cytoplasmic site of expression and concomitant use of vaccinia virus transcription factors. The cytoplasmic site avoids potential problems related to cryptic splice sites, processing, and nuclear-cytoplasmic transport. For example, the structural proteins of HIV-1 were expressed without the need for regulatory factors rev and tat (54,55). Mammalian cell codon optimization can increase expression, but is usually not required. The proteins made by recombinant vaccinia viruses are usually processed and transported in a manner similar to that occurring in uninfected cells. For example, N- and O-glycosylation (56), proteolytic cleavage (54,57), polarized membrane insertion

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(58), and nuclear transport (59) all occur. Biologically active enzymes, such as reverse transcriptase (55) and bacteriophage T7 RNA polymerase (55) and ion channel proteins (60) are made. Perhaps most striking is the expression of multiple genes by recombinant vaccinia virus leading to the assembly of infectious RNA virus particles (61–63).

RECOMBINANT VACCINIA VIRUS VACCINES Experimental Vaccines Vaccinia virus can infect most mammalian as well as avian species, making it a useful vector for studying the immune response to proteins of both human and veterinary pathogens. Recombinant vaccinia viruses are standard laboratory tools for defining protective antigens and fine-mapping immunogenic epitopes from infectious agents. Prophylactic live recombinant vaccinia viruses have protected animals from challenge with numerous viruses, bacteria, and parasites (64). In addition, poxvirus vectors expressing tumor antigens have been investigated for the immunoprophylaxis and immunotherapy of tumors in animal models (65–68).

Immunogenicity A particular advantage of vaccinia virus as a recombinant vaccine vector is its ability to induce immune responses similar to those elicited by viral proteins during the course of a natural infection. Consequently, cell mediated as well as humoral immunity is evoked (69). In some cases, antibody responses induced by recombinant vaccinia virus are as high as those induced by natural infection (70). Mice vaccinated with a recombinant expressing HSV glycoprotein D were still protected from a lethal challenge with HSV one year after vaccination (71), and macaques vaccinated with a simian type D retrovirus (SRV)-2 envelope glycoprotein recombinant were still protected against SRV-2 two years after vaccination (72). Route and dose of vaccinia virus are important in eliciting efficient immune responses. For replicating strains of vaccinia virus, dermal inoculation is considered to be the most immunogenic route in man, apparently reflecting a preferred site of replication of orthopoxviruses as well as the presence of a large number of antigen-presenting cells. Percutaneous vaccination with vaccinia virus produced significantly higher antibody titers and a longer duration of immunity than subcutaneous vaccination (73,74). Intramuscular vaccination of mice with a rabies virus nucleoprotein recombinant was substantially less immunogenic than intradermal inoculation (75). In primate species, there was a linear relationship between the size of skin lesions formed by vaccinia/respiratory syncytial virus (RSV) recombinants and titers of RSV neutralizing antibody (76). Intradermal vaccination of mice and hamsters with a vaccinia/influenza HA recombinant elicited mainly IgG, which prevented lower respiratory infection; intranasal inoculation induced secretory IgA production and prevented both upper and lower respiratory tract infections (77). Limited testing of oral smallpox vaccine in humans indicated that vaccinia is immunogenic when administered by this route. Successful oral vaccinations of domestic and wild animals with vaccinia/rabies glycoprotein recombinants (78,79) may be mediated by replication in tonsillar or buccal tissue (80). Furthermore, intrajejunal vaccination of mice with vaccinia-influenza recombinants leads to generalized mucosal immunity against influenza (81). Oral or intranasal routes of inoculation could potentially be exploited with other vaccinia-based veterinary or

human vaccines. For nonreplicating strains of vaccinia virus, such as MVA, a large inoculum size is needed, and therefore intramuscular or subcutaneous routes are usually used. Although disease protection correlates with vaccineinduced antibody titers in some cases, other vaccinia recombinants appear to protect by priming for immunoglobulin and/or inducing effector lymphocyte responses. In many cases, it is difficult to discriminate which of these responses is most important for protection without doing additional experiments. A vaccinia/Hepatitis B surface antigen (HBsAg) recombinant failed to stimulate protective titers of anti-HBsAg antibody in chimpanzees, but did protect against disease. Apparently, vaccinated animals were primed for accelerated antibody production and possibly cell-mediated immunity (82). Similarly, a recombinant expressing the bovine leukemia virus envelope glycoprotein failed to elicit neutralizing antibodies, but partially protected vaccinated animals presumably through induction of cell-mediated immunity (83). When a recombinant virus expresses only internal structural or regulatory proteins, which cannot induce neutralizing antibody, protection is attributed to cytotoxic T-cell responses. For example, mice vaccinated with vaccinia/influenza nucleoprotein recombinants developed lower respiratory tract infections after challenge with influenza A virus, but had reduced symptoms, and some were protected from death (84). The protection from influenza in mice vaccinated with vaccinia/influenza nucleoprotein appeared to be cytotoxic T lymphocytes (CTL) mediated, since vaccination was only effective in murine strains capable of generating a strong anti-nucleoprotein CTL response. Similarly, recombinants expressing the rabies virus nucleoprotein did not prevent illness, but prevented fatal rabies in dogs (85). A vaccinia recombinant expressing the murine cytomegalovirus immediate early protein pp89, an internal protein, which regulates gene expression, afforded partial protection against a lethal challenge (86). Depletion of vaccinated mice with antiCD8 antiserum abrogated protection, and adoptive transfer of CD8+ lymphocytes from vaccinia/pp89-primed animals into unvaccinated animals limited murine cytomegalovirus replication (86,87). In addition, CD4+ effector T cells may mediate the protective immunity induced by certain vaccinia/measles recombinants (88). Recombinant vaccinia viruses that express simian immunodeficiency virus (SIV) gag-pol proteins have provided protection in a monkey challenge model (89,90). Effector T cells may also play a central role in the antitumor response elicited by vaccinia recombinants. Although antibodies to tumor antigens are often present postvaccination, CTLs are likely mediators of tumor regression in a murine mastocytoma model (91). Several techniques have been developed to improve the immunogenicity of live recombinant vaccinia vaccines. Since the magnitude of the response may be dependent on the amount of foreign protein expressed, high levels of protein production can be desirable. This can be achieved through the use of strong natural or synthetic vaccinia promoters (31,32,92,93). Elimination of cryptic vaccinia transcription termination sequences from an HIV envelope gene boosted the level of HIV env production in infected cells (33). Moreover, the corresponding recombinant virus was more immunogenic than the vector containing the termination sequence, as determined by anti-gp160 antibody titers in vaccinated mice, and by enhanced lysis of infected human CTL target cells. A similar correlation between expression and immunogenicity was obtained using a recombinant MVA (94).

Chapter 33: Vaccinia Virus and Other Poxviruses as Live Vectors

Immunogenicity may also be enhanced by altering protein presentation on the surface of infected cells. Fusion of a secreted malaria blood stage antigen to the murine immunoglobulin G transmembrane anchor sequence resulted in surface expression of the protein and a greatly enhanced antibody response (95). A related strategy has been employed in which repeating epitopes of the malarial circumsporozoite protein (CSP) were fused to the ectodomains of the RSV glycoprotein G, thereby enhancing anti-CSP antibody titers in vaccinated animals (96). Elimination of proteolytic cleavage sites from the HIV env gene prevented release of gp120 from the env precursor gp160, and the resulting recombinant produced a stronger anti-gp120 antibody response in vaccinated animals, presumably by increasing the amount of surface-associated gp120 (97). The incorporation of additional helper T-cell epitopes in expressed antigens may also increase immunogenicity through enhanced recruitment and proliferation of B cells. A short peptide derived from the sequence of the neutralizing epitope of the viral capsid protein VP1 of foot-and-mouth disease virus produced a weak antibody response in cattle and pigs. However, fusion of the VP1 epitope to Hepatitis B core protein (HBcAg) led to a dramatic increase in antibody titers in animals vaccinated with the purified protein expressed by recombinant vaccinia virus and greatly enhanced virus neutralization (98). Inclusion of murine helper T-cell epitopes led to increased titers of anti-malarial antibodies in mice vaccinated with a CSP fusion construct (99). Protein targeting may also enhance immunogenicity. Fusion of antigen genes to endoplasmic reticulum or lysosomal targeting sequences may direct expressed proteins into intracellular compartments where processing for antigenic recognition is facilitated, boosting the immune response to viral and tumor-associated antigens. In one case, endoplasmic reticulum targeting was reported to increase CD8+ T cell recognition of tumor cells (91). Fusion of HIV gp160 sequences to the lysosomal targeting sequence LAMP-1 boosted the CD4+, class IIrestricted effector T-cell response to the expressed peptide, through enhanced transport of peptide into processing compartments (100). In addition, boosted antibody titers, lymphoproliferative, and CD4+ CTL responses followed immunization with a vaccinia LAMP-1/human papillomavirus E7 construct, as compared to the standard construct (101). Recombinant poxviruses that co-express certain cytokines or other mediators exhibit increased immune responses. Expression of interleukin (IL)-2 enhanced the serum IgG response to influenza nucleoprotein (102), and expression of IL-5 or 6 increased the secretory IgA response to influenza HA following intranasal vaccination (103). IL-2 co-expression with b-galactosidase enhanced activity against b-galactosidaseexpressing tumors in mice (104). Similarly, expression of granulocyte/macrophage colony-stimulating factor produced by recombinant avian poxviruses enriched the regional lymph nodes with antigen-presenting cells and acted as an immunoadjuvant (105). Expression of the CD28 ligand B7 by recombinant vaccinia viruses enhanced antitumor activity in mice by mediating a Th1-type T-cell response (106). Synergistic effects were reported for a triad of costimulatory molecules namely, B7-1, ICAM-1, and LFA-3 (107). Interferon-g, IL-12, IL-21 have also been reported to provide enhanced immune responses (108–110). Heterologous priming and recombinant poxvirus boosting greatly enhanced T-cell responses. This was first noted using a recombinant influenza virus as the prime followed by

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a recombinant vaccinia virus (111). This approach works particularly well using a DNA prime followed by a vaccinia virus or avipoxvirus boost (112–114). The generally accepted explanation is that immune competition between the many poxvirus proteins and the recombinant protein limits the extent of the immune response to the latter. However, if the animal has already made a primary immune response to the recombinant protein, then the recombinant vaccinia virus can preferentially boost this. The presence of maternal antibodies is a major obstacle to vaccination of infants with some attenuated viruses, such as measles, and also diminishes the immune response to recombinant vaccines. Passive administration of antiserum to influenza A (115) or RSV (116,117) abrogated both the desired antibody response and disease protection mediated by vaccinia-based influenza or RSV vaccines. Replication of the recombinant viruses and stimulation of antibodies to vaccinia proteins was unaffected, indicating that antibodies to the influenza or RSV proteins produced specific immune interference. Normal immune responses were restored after waiting for clearance of passively administered antibodies to measles virus in mice repeatedly vaccinated with vaccinia/measles recombinants (118). The inhibitory effect depends on the level of passive antibody, however, and a recombinant vaccinia virus provided significant protection of monkeys against measles infection (119). Changing the site of inoculation may partially overcome passive blockade of antigen, since intranasal administration of vaccinia/RSV vectors was significantly more immunogenic than dermal administration in cotton rats given anti-RSV antiserum parenterally (120). Existing immunity to the vector is a potential problem with any live recombinant virus, and this also holds true for vaccinia virus (71,121). Because of the cessation of smallpox vaccination in the early 1970s, individuals under 35 are generally vaccinia naive. Systemic immunity to vaccinia virus can be circumvented to some extent by administering the recombinant vaccine by a mucosal route (122). Repeated vaccination or priming with a DNA vaccine may also be useful. Because of their large genetic differences, immunity to vaccinia does not appreciably affect vaccination with avipox vectors.

Safety During the extensive use of vaccinia virus as a smallpox vaccine, adverse reactions in addition to the routine swelling and soreness at the site of administration and low-grade fever were observed. The most serious of these were progressive or disseminated infection in immunocompromised individuals, eczema vaccinatum, and postvaccinal encephalitis or encephalopathy in infants. The incidence of the latter was reported to vary with different vaccine strains, ranging from 1 in 2000 for the Copenhagen strain, to 1 in 200,000 or more for the New York City Board of Health (Wyeth) and Lister strains (1,123). There is a fear that adverse reactions would be even more prevalent now because of the high incidence of HIV, use of immunosuppressive drugs in transplant patients, and increased atopic dermatitis. Prior to the eradication of smallpox, more attenuated strains of vaccinia virus were made by serial passage in tissue culture (1). MVA, the attenuated strain most extensively tested in humans, was passed 570 times in cultured chick embryo cells and induces only a slight reddening at the site of inoculation (124,125). MVA has many gene deletions (126,127) resulting in

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an inability to replicate efficiently in human and most other mammalian cells (128–131). Further studies indicated that replication is blocked at a step in virus assembly and that early and late viral or recombinant protein synthesis occurs normally (132). Remarkably, recombinant MVA has been reported to induce as good an immune response in mouse and monkey models as replicating strains, though higher doses and boosting with a second vaccination may be needed (133,134). Avian poxviruses, which are naturally host-restricted in mammalian cells, also provide a safe vector system (135). With knowledge of the vaccinia virus genome sequence and functions of many genes (28), it has become possible to attenuate vaccinia virus by making specific deletions (136–138). Given the nearly 200 genes, there are many potential ways of attenuating vaccinia virus. NYVAC, a genetically engineered vaccinia virus with many of the same deletions as MVA, appears safe and immunogenic, although it has not been as extensively tested in humans (139,140). Coexpression of genes encoding certain immunomodulators attenuates live recombinant vaccinia viruses. IL-2 expression prevented generalized lethal vaccinia infection in immunodeficient athymic nude mice (102,141). This appeared to be mediated by elevation of natural killer cells and interferon-g activity in mice vaccinated with the recombinants (142– 144). IL-2 expression also attenuated recombinant vaccinia viruses in normal, immunocompetent rodents (102,145) and primate species (146,147) without significantly reducing vector immunogenicity. Similar results have been achieved with recombinant vaccinia viruses expressing interferon-g (148) and tumor necrosis factor-a (149). There needs to be some caution in inserting immunomodulators, however, since IL-4 increases poxvirus virulence (150,151). Increased safety of vaccinia virus might be achieved by altering the route of inoculation, although this could diminish immunogenicity (73,74). Subcutaneous, intramuscular, and oral routes reduce the risk of person-to-person spread of virus, but still leave the risk of progressive infection in the immunocompromised host. Given recent successes in the development of antiviral drugs, it should be possible to identify effective chemotherapeutic interventions for vaccinia virus (152). This could provide a ‘‘safety net’’ for the rare but serious adverse effects of vaccination. Certain DNA replication inhibitors such as Cidofovir and derivatives appear particularly promising (153–156). The drug ST-246, which inhibits the wrapping and transport of vaccinia virus, appears to be very potent and safe (157).

Veterinary Applications Both vaccinia virus and avian poxviruses are being used as vectors for veterinary vaccines. Successful oral vaccination of animals with a vaccinia virus rabies glycoprotein vaccine (158,159) led to its extensive field-testing as a wildlife vaccine. Live recombinant vaccinia vaccine–impregnated bait, scattered in areas where rabies is endemic, produced a protective immune response in the majority of animals. Raccoons (79) and foxes (78) were targeted in the United States and Europe, respectively. The vaccinia virus rabies glycoprotein vaccine contributed to the elimination of rabies in a red fox population in Belgium (160). A raccoon poxvirus recombinant expressing rabies virus glycoprotein has also been shown to be effective as an oral vaccine (161). A bait strategy has been proposed to control the fertility of wild or feral animal populations by using

live recombinant vaccines expressing anti-fertility immunogens such as b-chorionic gonadotropin (162). Recombinant vaccinia viruses that express vesicular stomatitis virus (163) or rinderpest (164) glycoprotein genes were shown to protect cattle against the respective pathogens. The rinderpest vaccine provides long-lasting protection, and because of its economical production and ease of administration, would be attractive for remote areas of the world (165). A recombinant capripoxvirus expressing the hemagglutinin protein gene has also been proposed as a rinderpest vaccine (166). Live recombinant vaccinia viruses have been shown to protect chickens against avian influenza (167) and Newcastle disease (168). Many other recombinant vaccinia viruses have been shown to be effective against pathogens in small animal models. Avian poxvirus recombinants are immunogenic in chickens and have been shown to protect against avian influenza virus (169–171), Newcastle disease virus (172), infectious bursal disease virus (173–175), and infectious bronchitis virus (176). Fowlpox virus vaccines against Newcastle disease virus and avian influenza virus have been licensed. Avian poxviruses are also immunogenic in non-avian species (9), and the demonstration that recombinant canarypox vectors protect canine distemper virus (177), feline leukemia virus (178), and rabies virus (179) has led to veterinary vaccines.

Clinical Applications Many recombinant poxviruses that express genes of human pathogens have been tested for immunogenicity in small animal models, and no attempt will be made to summarize them here. Instead, this section will be limited to selected candidate vaccines that have been used clinically or in nonhuman primates. Also largely omitted from this review are attempts to develop therapeutic cancer vaccines using poxvirus vectors (180). The demonstration that a recombinant vaccinia virus could protect chimpanzees against hepatitis B virus gave credibility to poxvirus vectors (82,181), though the existence of licensed vaccines against this disease was a factor in hindering its further development. Recombinant vaccinia viruses expressing Epstein Barr virus glycoprotein provided protection in marmosets (182,183) and in a small clinical trial (184). Phases I and II clinical trials have been carried out with a recombinant vaccinia virus Hantaan vaccine (185). After two doses by the subcutaneous route, Hantaan virus neutralizing antibodies were detected in 72% of vaccinia virus naı¨ve but in only 26% of vaccinia virus immune volunteers. For safety reasons, many subsequent studies have used poxviruses that are defective in replication in humans. Phase I trials of canarypox vectors expressing human cytomegalovirus glycoprotein B or phosphoprotein 65 were reported. The glycoprotein vaccine induced only a weak antibody response even after multiple inoculations but primed for a subsequent boost with an attenuated human cytomegalovirus (186); the phosphoprotein vaccine induced a cytotoxic T-cell response in all subjects (187). In another study, priming with a canarypox glycoprotein B vaccine and boosting with the subunit protein was not more immunogenic than priming and boosting with the subunit protein (188). A canarypox rabies glycoprotein vaccine elicited dose-dependent antibody responses that were in the protective range, though lower than that of the standard human diploid rabies vaccine (189). The immune responses in a phase 2 trials with a canarypox HIV vaccine were too low to warrant further clinical studies (190).

Chapter 33: Vaccinia Virus and Other Poxviruses as Live Vectors

A recombinant NYVAC expressing seven genes from all stages of the Plasmodium falciparum life cycle was tested in a phase I/II efficacy trial (191). Antibody responses were low but cell-mediated immunity was detected in most volunteers. After challenge with infected mosquitoes, only 1 of 35 was uninfected, but there was a small but significant delay to parasite development in the experimental group compared with the control. A prime-boost immunization regimen using DNA followed by recombinant MVA induced strong cellular immune responses against the P. falciparum TRAP antigen in chimpanzees (192). A multistage multiantigen DNA prime and canarypox boost malaria vaccine provided partial protection against P. knowlesi in rhesus macaques (193). Clinical studies are being carried out using prime boost combinations of MVA, DNA, and fowlpox vectors as candidate tuberculosis (194,195) and malaria (196,197) vaccines. Live attenuated measles vaccines are ineffective in the presence of maternal antibody, indicating a need for a vaccine that can be used during the first six months of life. Passive antibody studies in primates gave some hope that recombinant MVA expressing the measles F and HA genes may provide some protection under these conditions (119,198). An MVA expressing a truncated dengue type 2 E protein protected monkeys against a challenge (199). The most extensive preclinical and clinical investigations of recombinant poxviruses have involved those expressing HIV or related SIV genes. Recombinant vaccinia viruses were used to demonstrate CTL in HIV-infected humans (200,201). The first clinical studies, carried out with a recombinant vaccinia virus that expresses the HIV envelope gene gp160, provided evidence for safety, but were not highly immunogenic (202,203). A history of prior smallpox vaccination did not prevent an antibody response to gp160, although vaccinia naive subjects in general developed a more vigorous antibody response. Priming followed by boosting with recombinant purified protein, however, produced a strong anamnestic response, with the production of type-specific neutralizing antibody (204,205). Similar results were obtained using a recombinant canarypox expressing gp160 alone or with a gp160 boost (206). Other canarypox virus vectors expressed HIV gag, protease, pol and nef, as well as envelope and induced CTL memory in 61% of volunteers at some time during the clinical trial (207). Preclinical studies of MVA-based SIV and simian human immunodeficiency virus (SHIV) vaccines have shown some promise. MVA alone or preceded by a DNA boost induced high gag and env CTL levels in macaques (114,208,209). When given prophylactically, such vaccines significantly lower the viral loads and protect macaques against CD4 depletion and clinical illness due to SIV or SHIV challenge (114,134,210,211). In one DNA prime and MVA boost study, expression of env plus gag and pol provided more uniform protection than expression of gag and pol alone (212). Several phase 1 studies with recombinant MVA and NYVAC HIV vaccines have demonstrated immunogenicity (213–215). Because vaccinia virus vectors have the capacity to incorporate at least 25,000 bp of extra DNA (20), the concept of polyvalent vaccines is extremely attractive. Recombinant vaccinia viruses, which simultaneously express three or more foreign proteins, have been constructed (216,217). Besides enhancing risk/benefit ratios, polyvalency would circumvent the reduction in immunogenicity of live vectors accompanying revaccination. The logistics of developing such a polyvalent vaccine, however, may prove daunting.

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34 Replication-Defective and Competent Adenovirus Recombinants as Vaccine Vectors Marjorie Robert-Guroff Vaccine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

Gary J. Nabel Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

John W. Shiver Department of Vaccines and Biologics Research, Merck Research Laboratories, Merck and Company, West Point, Pennsylvania, U.S.A.

ADENOVIRUS BIOLOGY AND VECTOR DEVELOPMENT FOR GENE DELIVERY AND VACCINES Adenoviruses (Ads) have a long history of use as vectors for gene delivery, in part due to their ability to infect nondividing as well as dividing cells and to express transgenes at high levels. Ads can be grown to very high titers, making them suitable for large-scale manufacturing and clinical development. Their promise for gene therapy applications provided an impetus for a detailed understanding of the virology and molecular biology of the virus, particularly Ad serotypes 2 and 5. The use of Ad vectors as vaccines has been more recent: studies in animal models and humans have shown promise and are reviewed in this chapter. Recombinant Ad (rAd) vectors have been designed to deliver a myriad of vaccine antigens, including gene inserts encoding proteins of DNA viruses, such as Epstein-Barr virus (1), Herpes simplex virus type 1 (2), pseudorabies virus (3), and hepatitis B virus (4), and doubleand single-stranded RNA viruses, such as rotavirus (5), vesicular stomatitis virus (6), rabies (7,8), measles (9), respiratory syncytial virus (10), parainfluenza virus (11), tick-borne encephalitis virus (12), Ebola (13), and Marburg (14) viruses. They have also been exploited for delivery of HIV and SIV antigens (15–17) and currently represent one of the most promising strategies for AIDS vaccine development. To understand why this is so, one needs to understand the biology of the virus and key characteristics (Fig. 1). Ads are double-stranded DNA viruses with high genetic stability, exhibiting no mutations or insert deletions after multiple rounds of replication in vitro. Further, they do not integrate into the genome of the infected host, and consequently present less of a safety concern for gene delivery, as there is little risk of insertional mutagenesis and expression of potentially toxic or deleterious gene products is finite. Because they are nonenveloped, they are physically stable and can withstand lyophilization, suggesting a convenient means for storage,

transport, and vaccine formulation relevant to global distribution. The primary targets of Ad infection are epithelial cells that line the respiratory tract and gut, key mucosal inductive sites. Ad displays the ability to induce mucosal immune responses, believed critical for mediating protective efficacy against infectious agents such as HIV, whose prime infection route is across the rectal and genital mucosa. In addition, many Ad serotypes readily infect dendritic cells (DCs), whose specialized ability to synthesize and present antigens is likely responsible for their potent immunogenicity. Ads, in fact, have been termed ‘‘nature’s adjuvants’’ (18) because of their ability to upregulate costimulatory molecules and elicit cytokine and chemokine responses following target cell infection. These attractive features have pushed Ad vectors to the forefront as vehicles for vaccine delivery. However, a significant concern in all rAd vaccine approaches is whether prior immunity to the vector, or anti-vector immunity induced as a result of immunization, will minimize the effectiveness of Ad vaccination. This concern arose principally as a result of gene therapy studies in which rAds were administered repeatedly at high dosages to maintain persistent expression of the therapeutic transgene. The result was development of vector immunity, which ultimately precluded continuous effective treatment (19,20). The extent to which such vector immunity will impact the utility of Ad-recombinant vaccines is currently not known and depends in part on the number and dosages of immunizations necessary to elicit the desired level of adaptive immunity. In principle, vaccination regimens should require lower doses of immunogen, administered infrequently to elicit immune memory responses. Nevertheless, several approaches have been exploited to circumvent this potential problem and will be discussed in this chapter. These alternatives include the use of alternate serotypes, Ad vectors of nonhuman origin, and chimeric Ad vectors in which neutralizing epitopes on the virion surface have been substituted with epitopes representing rare Ad serotypes.

Chapter 34: Ad Vaccine Vectors

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Figure 1 Structure of the Ad shaft and interaction with putative receptors on DC that facilitate gene delivery and subsequent antigen presentation to stimulate adaptive immunity. (A) Location and structure of the viral shaft on the adenovirus particle. (B) Schematic representation of the interaction of the Ad fiber with a putative receptor on immature DC that facilitates gene delivery and subsequent differentiation and enhanced antigen presentation. Abbreviations: Ad, adenovirus; DC, dendritic cell.

Finally, most Ad vectors exploited for vaccine development to date have been non-replicating, engineered with a deletion in the E1 region genes necessary for viral replication. This feature enhances the safety of the vector, but has other consequences, such as the high doses necessary to elicit potent immunity. An alternative is the use of replication-competent Ad vectors. Wild-type replicating Ad4 and Ad7 have a long history of safe use in the U.S. military and provide the basis for pursuing this approach. Recently, replication-competent Ad vectors have shown promise in the HIV vaccine field (21,22) Although they have not been developed to the extent of replication-defective vectors, which are currently in phase IIb clinical trials (23,24), they are being advanced to human phase I studies. Some of the most effective vaccines to date have been based on live attenuated, replicating organisms. Examples include not only Ad, but also vaccines against smallpox, rabies, anthrax, Bacille Calmette-Gue´rin, yellow fever, poliovirus, measles, varicella, rotavirus, and rubella (25). Important issues for replication-competent Ad vectors remain those of safety, dependent in part on the route of immunization, and immunogenicity in humans. In this chapter, we will summarize the advantages and disadvantages of replicating and nonreplicating Ad vectors and present the current state of the field.

REPLICATION-COMPETENT rAd VECTORS FOR VACCINES Replication-competent Ad-recombinant vaccines have an established record of efficacy and safety in humans on the basis of wild-type Ad4 and Ad7 vaccines. Because they are replicationcompetent and there is limited preexisting immunity to Ad4 and Ad7 in the population, relatively low doses elicit protective immune responses. Studies in nonhuman primates have shown that replicating rAds elicit sustained and potent immune responses that confer protective efficacy in lentiviral challenge models.

The immunogenicity of live, replicating vaccines must always be weighed against safety considerations. For replication-competent Ad-recombinants, the prototype Ad4 and Ad7 wild-type vaccines, routinely administered to military recruits from 1971 to 1996 (26) provide a strong safety record. These live, oral vaccines, safely administered to over 10 million people, were highly effective in controlling acute respiratory disease in the military setting. The vaccines were fully licensed, but never recommended for general use as a civilian need was not documented. Following cessation of production of Ad4 and Ad7 vaccines in 1996, outbreaks of Ad4- and Ad7-induced acute respiratory disease reappeared in the barracks setting, and the need for resuming the military vaccination program became apparent. Currently, manufacture of new vaccine lots is underway. A key safety feature of the replicating wild-type Ad4 and Ad7 vaccines is their formulation for oral delivery as entericcoated capsules. The enteric coating prevents dissolution of the capsule in the acid environment of the stomach, and allows delivery to the intestine, where the capsule disintegrates in the neutral pH environment. The oral delivery allows the vaccine to bypass the upper respiratory tract, thus preventing disease. Nevertheless, the vaccine virus causes an enteric infection that is immunogenic and highly effective. Further, the lyophilized virus within the capsule provides a stable, easily stored product. Vaccine administration is readily accomplished, with no needles or special equipment required. The intranasal administration route has been explored for vaccines intended to elicit mucosal immunity (27). Studies in chimpanzees have suggested that an intranasal replicationcompetent Ad-recombinant vaccine might be more immunogenic and efficacious than an oral one (28). However, this route is associated with greater safety concerns. Following high-dose (1010 viral particles) intranasal administration to mice of a replication-defective Ad, virus was detected in the olfactory bulb (29). Natural Ad infections transmitted via the upper

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Table 1 Features of Replication-Competent and ReplicationDefective rAd Vaccine Vectors Shared characteristics Ease of growth to high titer High level expression of inserted genes Genetic and physical stability Infection of dividing and nondividing cells Lack of integration Infection of dendritic cells Induction of mucosal immunity Additional features: replication-competent adenovirus vectors Lower doses needed to elicit potent immunity Prolonged expression of inserted genes Induction of cytokines and costimulatory molecules as natural adjuvants Additional features: replication-defective adenovirus vectors Greater safety Ability to accommodate larger inserts Flexibility in chimeric vectors for avoiding preexisting immunity

respiratory tract are not known to cause brain disease, and intranasal administration of replication-competent Ad4 vaccines to people is safe at low doses (30). Nevertheless, cautious testing of replication-competent rAd and replication-defective rAd vectors via this immunization route will be required (Table 1). Safe dosages of oral Ad4 and Ad7 vaccines have been established, ranging from 104 to 107 TCID50 in Ad-seronegative individuals (31–33). Similarly, a replication-competent Ad7hepatitis B surface antigen recombinant was safely administered in an enteric-coated tablet at a dose of 1.6  107 pfu (34). Safe, well tolerated intranasal doses of the Ad4 wild-type vaccine range from 2  105 pfu in Ad4-seropositive individuals and 4  104 pfu in Ad4-seronegatives (30). The established doses of oral, wild-type Ad4 and Ad7 vaccines are safe and effective. The relatively low dose needed to elicit protective immunity, compared with a replicationdefective vector, provides for ‘‘dose sparing,’’ which reduces the production requirements and lowers the cost of manufacturing. For vaccines needed for global distribution to tens or hundreds of millions of people, such as those designed to protect against HIV/AIDS, this factor could allow the generation of sufficient vaccine to meet the anticipated demands. A safety concern for any replication-competent vector is the possible transmission of the vector. The oral wild-type Ad4 and Ad7 vaccines, although shed in stool specimens, are not transmitted by casual contact such as occurs between vaccinated and unvaccinated military recruits in close contact during basic training (35). Transmission of these vaccines has been seen only in the close family setting between 70% of couples tested, suggesting that intimate contact is a factor (36). Mother to child transmission or transmission from a vaccinated child to a parent or sibling occurred with a lesser frequency, ranging from 2% to between 10% and 20%, respectively (37). Transmission studies of intranasal Ad vaccines have not been so extensive. However, no transmission was observed between jointly housed vaccinees and controls following administration of Ad4 vaccine to either Ad4-seronegative or -seropositive individuals (30). The selection of Ad4 and Ad7 as vaccine vectors is based not only on the history of the wild-type vaccines but also on their limited preexisting immunity in the population. Following

cessation of the military vaccine program in 1996, 66% and 73% of new recruits lacked Ad4 and Ad7 protective antibodies, respectively (38). Together, 88% lacked protective immunity to one or both of the viruses, suggesting that administration of the vaccines together, as has been safely done (31), would protect the vast majority of vaccinees. The need for resuming the military vaccine program substantiates the lack of protective immunity to Ad4 and Ad7 in young adults. With this extensive history of safe and effective use, development of live-replication competent Ad-HIV recombinant vaccines was initiated in a dog model, using Ad4-, Ad5-, and Ad7-HIV envelope (Env) recombinant priming and Env protein boosting (39) to induce high titered neutralizing antibodies. The sequential use of vectors of different subtypes circumvented vaccine-elicited immunity that might dampen induced immune responses following booster administrations of the same vector. Humoral, cellular, and mucosal immune responses were shown to be induced by a similar prime-boost approach in the chimpanzee model (28,40). Durable immune responses, protective efficacy against low- and high-dose HIV challenges, and protection against a high-dose, heterologous, primary HIV isolate were demonstrated (41–43). Pathogenic isolates could not be used in chimpanzees, and HIV challenge stocks titered for mucosal administration were not available. Therefore, further development of the approach was carried out using the SIV rhesus macaque model and an Ad5 host range mutant (Ad5hr) vector able to replicate in monkey cells (44). In subsequent preclinical studies, induction of cellular, humoral, and mucosal immune responses was demonstrated and increasing levels of protection were observed against mucosal SIV challenges administered intravaginally or intrarectally as the recombinant vaccines were targeted to additional SIV gene products (45–48). Recently, priming of rhesus macaques with rAd5hr expressing the SIVenv/rev genes plus SIVgag, SIVnef, or both, followed by boosting with Env protein subunits, elicited potent protection against intrarectal challenge with pathogenic SIVmac251 (21). A subset of 39% of the vaccinated animals displayed no viremia or viremia around the threshold of detection. Reduction in acute-phase viremia was associated with antiEnv-binding antibodies, and reduced chronic-phase viremia with cellular immunity to Env and Rev. Of interest, the binding antibodies mediated antibody-dependent cellular cytotoxicity (ADCC), also correlated with decreased acute viremia (49). Seventy-three percent of these highly protected macaques subsequently resisted a second challenge administered a year later without any intervening immunization, thus demonstrating durable protection (22). Additional studies have been carried out in a permissive species, the chimpanzee, in which use of a host range mutant was not necessary, and recombinants based in Ad vectors intended for human use could be evaluated. The immunogenicity of a regimen combining priming with replication-competent Ad5 and Ad7 recombinants expressing the HIVMNenv/rev genes and boosting with oligomeric HIVSF162 gp140DV2 was compared with a similar regimen in which priming was accomplished with replication-defective Ad5 and Ad7 recombinants (17). At the same or lower dose, the replicating recombinants better elicited HIV-specific cellular immune responses, including a greater frequency of HIV-Env-specific interferon-gamma (IFN-g)-secreting peripheral blood cells and better priming of T-cell proliferative responses, though stronger immune responses might have been elicited with higher doses of

Chapter 34: Ad Vaccine Vectors

replication-defective rAd vectors. Enhanced humoral immunity was manifested by higher anti-Env-binding and -neutralizing antibody titers. The vaccination strategy elicited neutralizing antibodies against a number of heterologous primary HIV isolates, more frequently in animals primed with the replicating recombinants. The enhanced immunogenicity may be attributed to a greater effective dose following in vivo replication, and the elicitation of cytokines and costimulatory molecules, which serve as natural adjuvants as the host responds to a subclinical infection. More recent studies have made use of SHIV models, which allow investigation of vaccine strategies targeting the HIV Env, considerably different than the SIV Env. Priming with Ad-HIV recombinants encoding Tat and Env elicited greater protection against a pathogenic SHIV89.6P challenge than a multigenic vaccine regimen with priming by Ad-HIV tat, env, gag, and nef recombinants (50). Significantly higher titer antibody to both Tat and Env was observed in animals immunized with the Tat plus Env vaccine regimen compared with the multigenic regimen, though the mechanism of protection is unclear. Further, evidence that a protein boost contributes to the overall vaccine efficacy via reduction in acute viremia has recently been obtained (51). These studies have provided preclinical data in support of phase I human trials. An Ad4-HIV recombinant expressing the env gene and formulated for oral administration is being produced, and manufacture of a similar Ad7 recombinant to allow testing of the sequential vaccine strategy is planned. Replication-competent Ad-recombinant vaccines may be more immunogenic in people than in nonhuman primates because of differences in replicative capacity between species. Phase I trials will address the relevant safety and immunogenicity questions related to replication-competent Ad recombinants and determine the extent to which the approach merits further development.

REPLICATION-DEFECTIVE rAd5 VACCINE VECTORS The use of replication-defective Ad5 as a vaccine vector followed the initial development of rAd5 for experimental gene therapy applications. These vectors are rendered replicationdefective by removing the E1 gene, which is required for both replication and downstream gene expression. For cell culture of rAd, E1 function is provided in trans using E1-transduced cell lines such as 293 and PER.C6TM cells. E3, which is nonessential for viral replication, is also removed in some rAd vectors to provide greater capacity for both regulatory elements and genes encoding antigens. One of the primary drivers for development of vectors for vaccines was the increased awareness of the importance of cellular immune responses for containing infections both following natural infection as well as for developing protective immunity by live viral vaccines. A variety of different approaches were tested largely in preclinical animal models to determine the best means for eliciting cellular immune responses, particularly those mediated by CD8+ T lymphocytes. These approaches fell primarily to one of two categories: (i) adjuvanted peptides or recombinant proteins, especially virus-like particles and (ii) gene delivery vehicles designed to encode antigens derived from pathogens and express these antigens in vivo following injection into organisms. Gene-based vaccination thus induced immune responses against the antigen in a manner similar to that of the

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natural microbe. Numerous reports have been published describing the use of plasmid DNA, bacterial vectors such as salmonella or shigella, and viral vectors based on alternative viruses, including poxviruses [e.g., vaccinia, modified vaccinia Ankara (MVA), and canarypox], alphaviruses (e.g., Venezuelan equine encephalitis virus and Sindbis), and adenoviruses (both live and replication-defective) as vaccine vectors. Such studies demonstrated that rAd vectors were among the most potent means for eliciting cellular immune responses in both rodents and nonhuman primates. In particular, head-to-head comparisons of rAd5 vectors with plasmid DNA and pox viruses using HIV-1 or SIV Gag antigens showed that rAd5 vectors elicited both cellular and humoral immune responses in nearly all animals at least several fold greater than DNA- and pox-based vaccines (15,52). In the study using SIV Gag as antigen, rhesus monkeys were challenged with pathogenic SHIV89.6P. The rAd5 vector-immunized animals were able to control both viremia and CD4 cell depletion, while both the MVA and plasmid DNA vaccinees were unable to control infection or disease progression (15). In fact, control of infection showed a significant inverse correlation to the magnitude of CD8+ T-cell response elicited by immunization so that the rAd5 vector vaccinees, having the highest levels of cellular immune responses following immunization, exhibited the lowest levels of viremia after challenge (15,53). On the basis of these data, Merck began clinical studies of both DNA and rAd5 vector vaccines encoding HIV-1 Gag in normal, HIV-negative adult humans in 1999 and 2000, respectively. While both vaccines were well tolerated at all doses, the rAd5 vaccine was clearly superior to plasmid DNA both in terms of proportion of vaccinees that generated an immune response as well as magnitude of response. The response levels as measured by IFN-g enzyme-linked immunospot (ELISPOT) assays were comparable with those produced in rhesus monkeys for the rAd5 vaccine. Despite these encouraging data, it was clear that preexisting neutralizing antibodies against Ad5 significantly affected the immunogenicity of this vaccine. There was a clear dose to baseline neutralizing anti-Ad5 titer relationship that reduced both the magnitude of response and proportion of responders. Immune responses were largely abrogated at the lowest doses tested (1e8 to 1e9 vp) at Ad5 titers >200. However, most vaccinees developed an immune response at the highest doses tested (1e10 to 1e11 vp), although the proportion was still somewhat lower than for individuals that had relatively low baseline Ad5 titers. Subsequently, Merck and the HVTN tested a trivalent rAd5 vector vaccine comprising individual vectors expressing HIV-1 gag, pol, or nef. This study also showed the rAd5 vector vaccines to be well tolerated and immunogenic for each of the three antigens. Interestingly, preexisting Ad5 antibodies appeared to have a lesser effect on development of immune responses in vaccinees in this study than in the monovalent rAd5 vector Gag trials. On the basis of these data, Merck and the HVTN initiated a ‘‘proof-ofconcept’’ efficacy clinical trial using the trivalent rAd5-Gag, -Pol, -Nef vector vaccine. This trial was designed to test for both prevention of infection and reduction of virus load following infection, the latter being the effect of immunization that was observed for this vaccine in nonhuman primates challenged with SHIV and SIV. The trial was also designed to test vaccine efficacy both in populations having low and high baseline Ad5 titers as well as populations exposed to different clades of virus. The low titer Ad5 seropositive group who were exposed largely to viruses more closely related to vaccine antigens

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(clade B) were intended to give the clearest indication of whether the biological basis for the vaccine is valid, the high titer Ad5 seropositive background to determine whether preexisting immunity to Ad5 would limit the vaccine efficacy, and the vaccine group in southern Africa was meant to address the impact of viral antigen divergence from vaccine antigens (clade C vs. clade B). Results from these studies have since shown that the vaccine had no effect on decreasing viral load (54). In addition, there was a trend towards increased acquisition in Ad-seropositive subjects, suggesting that the vaccine did not confer protective immunity in this subgroup. This information has informed the development of vaccines that can further improve upon the breadth and magnitude of immune response such as using viral vectors that have limited pre-exposure in humans and by the addition of more antigens. Several approaches have been developed to increase the potency of rAd5 vector vaccines. Most of these are variations of heterologous ‘‘prime-boost’’ immunizations in which one type of vaccine modality is used to initiate an immune response and a second modality to provide a boost. Some of the most common, and successful in preclinical studies, of these include plasmid DNA prime with rAd vector boost, rAd vector prime with pox virus boost, or rAd vector prime boost using different serotypes of adenoviral vectors (55,56). Each of these approaches has been shown to elicit enhanced antibody and T-cell responses in rodents and nonhuman primates compared with using single modalities alone. Interestingly, some of these combinations were ineffective when used in the opposite sequence. For example, DNA boost after viral vector prime and pox virus vector prime followed by rAd vector boost did not show enhancement of immune responses over individual components used alone. In clinical studies, it has been difficult to show enhanced immune responses using these types of heterologous prime-boost immunization regimens. Vaccine trials in which Merck vaccines were tested did not find enhancement of responses using DNA/rAd5 vector or rAd5 vector/ canarypox prime-boost regimens compared with regimens using a similar number of immunizations with rAd5 vectors alone. However, clinical studies conducted by the NIH Vaccine Research Center suggest that DNA/rAd5 vector prime-boost does show an additive or synergistic effect compared with either vaccine used alone, particularly when Env is included as an immunogen. Such studies will need further exploration in humans but certainly highlight some of the challenges associated with attempting to use animal studies to provide guidance for human clinical trials. While the application of these new vaccine approaches has been largely for development of a protective vaccine against HIV-1 infection as well as better influenza vaccines, these technologies have been tested against other diseases as noted above. Typically, rAd5 vector vaccines were able to induce both cellular and humoral immune responses in rodents and/or nonhuman primates that in some cases provided protection against challenge. Several examples of successful immunization of nonhuman primates using rAd vector vaccines are especially noteworthy. Sullivan et al. reported that rAd5 vector immunization provided protection against Ebola virus infection of cynomolgus rhesus monkeys (13). Folgori and coworkers (57) showed that chimpanzees primed with DNA encoding hepatitis C virus (HCV) NS protein followed by rAd6 vector boosting were able to significantly attenuate viremia following HCV challenge and cleared virus much more rapidly than control animals. The combination of DNA/rAd vector prime boost has also been reported to break tolerance to

self-antigens associated with cancer in both rodents and nonhuman primates (58).

ALTERNATIVE ADENOVIRUS VECTOR SEROTYPES While rAd5 vectors have shown impressive immunogenicity in animal models and in early human trials, natural immunity to Ad5 could potentially affect its efficacy as a vaccine vector. Ad5 seroprevalence varies according to geographic location. In the United States, seroprevalence is approximately 50%, while in certain countries in Africa, up to 90% of individuals may possess high titers of neutralizing antibodies (59–63). The extent to which this immunity may impact the effectiveness of rAd5 vaccines is not completely known. Evidence that preexisting immunity can reduce immunogenicity comes from studies in mice (64,65), rhesus monkeys (52), and possibly in humans (66,67). At the same time, this effect may vary depending on the strength of the immunogen, the rAd5 vector viral particle dose administered, and the magnitude of the baseline Ad5 neutralizing antibody titer. For example, a reduction in the Gag-specific response was observed in a first-generation rAd5 vector encoding Gag during the Merck/HVTN AIDS vaccine trials. This inhibition was most pronounced at relatively low Ad5 doses and for the highest baseline Ad5 titers and was mostly offset, but not completely, at the highest doses tested (1e10 to 1e11 vp) (Fig. 2). To address this potential problem and to develop additional vectors for different prime-boost combinations, alternative serotype Ad vectors have been generated. Among these vectors are strains that show lower seropositivity among various populations (62,63). Those that have undergone further analysis include representatives from subgroups B and D, including Ad11, 35, 26, and 48. Ad26 vectors have recently undergone further development and appear to show promising T-cell immunogenicity in animals. Rare serotype Ad vectors have generally proven less immunogenic than Ad5 vectors in mice and rhesus monkeys (56,68–71). The seroincidence of rAd35 is among the lowest in humans, and rAd35 vectors therefore advanced more quickly into human studies, where it is under study for tuberculosis, malaria, and HIV. Human trials conducted by the Vaccine Research Center, NIAID, NIH, and the HIV Vaccine Trials Network have recently begun to assess the immunogenicity and safety of a replication-defective Ad35 vector encoding an HIV antigen, clade A Env. The goal of these phase I studies is to establish the relative efficacy of rAd35 vaccine vectors, which appear lower than Ad5 vectors in animal studies (69,70). In particular, it will be important to determine whether the rAd35 vector will elicit immune responses comparable or greater than rAd5 vectors in Ad5 seropositive individuals. These studies will also examine the relative efficacy of DNA/rAd35 vectors and rAd35/rAd5 vector prime-boost combinations. The specificity, immunogenicity, and reactogenicity of rAd vectors are determined by their external coat proteins. The major target of Ad-neutralizing antibodies is the hexon (71). For this reason, emphasis has been placed on constructing chimeric Ad vectors in which these components have been modified selectively to avoid naturally circulating neutralizing antibodies directed to these gene products. Among the approaches, the ability to introduce heterologous hexon sequences into the exposed external regions of the Ad5 hexon has enabled the development of a novel Ad5-like vector, which is largely insensitive to preexisting neutralizing antibodies (72). This vector can be produced in rAd5 packaging cell lines and shows immunologic

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Figure 2 Approaches to the development of alternative rAd vectors that evade preexisting immunity. Alternative strategies to the creation of Ad vectors with minimal reactivity to natural anti-Ad immune sera by (A) generation of chimeric rAd vectors, exemplified here with various components between Ad35 and Ad5, though such chimeras can be prepared in principle between any two Ad vectors; (B) replacement of the hypervariable region of the hexon with novel amino acid sequences to evade neutralizing antibodies reactive with this region; and (C) the use of naturally occurring Ad from rare human serotypes or from other species, including sheep or nonhuman primates. Abbreviations: rAd, recombinant adenovirus; Ad, adenovirus.

properties similar to rAd5 vectors. It is a possible vector for human studies that has performed well in studies (71) and may undergo further clinical evaluation for AIDS vaccines. The mechanistic basis for alternative properties of these adenoviruses with respect to immunogenicity and reactogenicity is not fully understood. In part, these activities may be mediated by specific receptors engaged by the adenoviral vectors. The immunogenicity of rAd5 vectors is independent of the coxsackievirus and adenovirus receptor (CAR)-binding receptor and largely dependent on a heparin-sensitive interaction through the Fiber shaft, although a small quantitative increase of the CAR domain may contribute to this effect. In contrast, Ad35 utilizes the CD46 receptor, which in other viruses has been shown to induce a more Th2/immunosuppressive response, as is also seen with the measles virus. Further analysis of the mechanism of attachment and entry of the alternative serotypes will prove useful. The Ad5 fiber can also contribute to neutralization sensitivity (70,73), though less than hexon. In addition, it has recently been shown that the reactogenicity of Ad5 is mediated by the fiber shaft, which may also confer targeting to DCs. The

construction of alternative chimeric fibers, together with adenovirus serotypes of lower prevalence such as Ad35, provides a mechanism by which to enhance immunogenicity and also to evade preexisting immunity. It remains unclear whether the problem of increased reactogenicity of the Ad5 vector can be circumvented by using this approach. In addition to rare serotypes and the chimeric rAd vectors, Ads derived from nonhuman species have been developed as vaccine and gene transfer vectors. Among them, ovine, bovine, porcine, and chimpanzee adenoviruses have been developed (74–77). In contrast to human adenoviruses, where toxicities can be understood in part through natural human infections with known disease symptomatology and long-term sequella, these issues are unknown for viruses derived from other species. Safety concerns with respect to the animal viral vectors will therefore likely require sufficient safety testing for regulatory approval (Table 2). In addition to their value in evading host immunity, alternative adenoviral vectors can also serve to enhance immune responses by using alternative prime and boost approaches. For example, although Ad35 vectors induce a

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Table 2 Strategic Considerations for Development of Novel Chimeric and Rare Serotype rAd Vectors 1. Immunogenicity-improved delivery of foreign proteins to antigen-presenting cells 2. Evasion of natural anti-vector immunity 3. Reduction of reactogenicity, e.g., improved safety 4. Development and enhanced potency of novel recombinant adenovirus prime-boost combinations

12.

13.

14.

lower magnitude immune response, they appear to serve better to prime immune responses. Ad5 appears to be particularly effective in boosting immune responses. The use of DNA priming followed by adenoviral boosting was initially described in an Ebola virus challenge model where previous vaccine approaches had proven unsuccessful (78). This latter approach using DNA priming followed by rAd vector boosting has now been applied to other disease models, particularly to lentiviral vectors, where prime-boost combinations have proven to confer partial protection in rhesus macaques against SHIV89.6P (15,16), SIVmac239 (56), and SIVmac251 (79–81). Thus, the alternative Ad serotypes, whether human or derived from animal species, can both help to circumvent prior humoral immunity and stimulate optimal prime-boost combinations.

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35 RNA Virus Replicon Vaccines Nancy L. Davis and Robert E. Johnston Carolina Vaccine Institute, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, U.S.A.

INTRODUCTION Expression systems based on single-stranded RNA viruses offer exciting prospects for their application as vaccines. The development of cDNA-based genetic systems for a substantial number of these viruses has made possible the directed manipulation of RNA virus genomes, the discovery of substantial genomic plasticity in these virus systems, and their reconfiguration as vaccine vectors. Pioneering work with infectious clones of poliovirus (1), positive strand RNA plant viruses (2,3), and rabies virus (RV) (4) was key in guiding subsequent development of analogous reverse genetic systems for other RNA viruses and vaccine vectors derived from them. Single-stranded RNA virus vectors can be divided conveniently into those based on positive or negative stranded genomes. Positive stranded genomes have the same sequence as mRNA, and their genomes are infectious when introduced alone into a cell by any one of a number of transfection methods. For genetic manipulation of a positive strand RNA genome, cDNA clones of the viral RNA are linearized downstream of the viral sequences, and in vitro transcription results in the production of positive strand RNA replicas of the viral genome which also are infectious. Negative stranded RNA genomes carry their genetic information in the complementary sense relative to mRNA. Therefore, successful virus propagation requires that the viral replicase/transcriptase enzymes required in the earliest stages of intracellular replication be introduced into the cell along with the genome. In systems designed for reverse genetics of negative stranded viruses, in vitro transcripts from cDNA clones (usually transcripts that are the positive sense complement of the genome) are introduced into cells where the replicase proteins are being expressed from a second expression system (often the vaccinia T7 system). With either positive or negative sense RNA genomes, the systems used to regenerate infectious viruses from cDNA clones have also been employed to add immunizing genes from target pathogens to the viral genomes. This results in a fully infectious and propagation competent virus vector, which expresses an exogenous immunogen in addition to a full complement of viral proteins in the successive cells it infects (Fig. 1A.1). A second iteration on this theme is the substitution of an immunogen gene for one or more of the structural protein genes of the vector virus. The resulting RNA genome is selfreplicating inside an appropriate cell, thus the term ‘‘replicon.’’ The replicon expresses the immunizing gene and can be packaged into ‘‘replicon particles’’ when the structural proteins are produced in trans within the same cell (Fig. 1B). When

inoculated into an animal or human, the replicon particles target to those cells normally infected first by the complete virus, the replicon genome expresses the immunizing gene, but the infection cannot be propagated to additional cells due to the absence of the structural protein genes from the replicon genome. Alternatively, a positive sense replicon can be transcribed, or launched, from a DNA vaccine in vivo. Thus, RNA replicons delivered as replicon particles or launched from DNA constructs can provide safe and effective immunization against a variety of pathogens. Table 1 provides a list of RNA virus vectors discussed in this review.

VACCINE VECTORS DERIVED FROM POSITIVE STRAND RNA VIRUSES Propagation Competent Vectors Propagation competent vectors express an immunizing gene in the context of a complete virus genome. For instance, a second copy of an alphavirus subgenomic mRNA promoter can be inserted into the genome for expression of an immunizing transgene (5–9). Coronaviruses express their genes from a 30 co-terminal nested set of mRNAs. An exogenous immunizing gene may be inserted between native coronavirus genes, and the gene will be expressed if the cis-acting intergenic region signals, necessary for transcription of mRNA, are included at its 50 end (10). In picornavirus propagation competent vectors, short, foreign epitope sequences have been substituted for external loops on the poliovirus capsid structure (11), or an immunizing gene has been placed either upstream of the major capsid genes or at the P1/P2 junction, maintaining the genomic open reading frame (ORF; 12–14). Crotty et al. (15,16) overcame the inherently limited insertion size in such vectors by expressing a library of relatively short overlapping coding sequences from genes many times larger than the insertion maximum. Flaviviruses have been modified in a manner analogous to picornaviruses (17,18). In addition, the 17D yellow fever vaccine, as well as attenuated dengue fever virus subtypes, have served as a backbone for flavivirus chimeras in which the prM and E genes of a flavivirus to be vaccinated against are exchanged for the same genes in one of the vaccine virus backbones (19–21) (Fig. 1A.2). The goal is to take advantage of the attenuating mutations embedded in the vaccine backbone to create alternative live virus vaccines for the agents supplying the exogenous prM and E genes. Human clinical trials of this concept are underway (Japanese encephalitis) (22–24).

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Figure 1 RNA vector genomes and packaging helpers. (A) Propagation-competent virus vectors have all the genes required for replication of the RNA genome and production of progeny virus. 1. Insertion site—region(s) of the genome into which a heterologous gene sequence can be introduced, and from which it can be expressed while maintaining a viable virus genome. One or more inserted genes may be added between vector genes or may replace the coding sequence for a nonessential protein. This group also includes vectors carrying a structural protein gene that has been altered by the insertion of a small heterologous sequence. 2. Chimeric vector—results from the substitution of the structural genes of the original virus, including major immunogenic components, with those of a second virus. (B) Nonpropagating virus vectors can undergo only a single round of replication because they lack the genes required for morphogenesis and progeny virion release. 1. RNA replicon—includes the genes essential for RNA replication and gene expression and the heterologous antigen gene, inserted in a way that allows both its expression and replicon RNA replication. Replicon RNA can be supplied as an in vitro RNA transcript or launched from a cDNA. 2. Helper genes—required for production of single-cycle virus vector. Supplied in trans by a helper virus, a packaging cell (constitutive or inducible nuclear genes), or helper genes on a transfected DNA or RNA.

Advantages and Disadvantages of Propagation Competent Vectors Propagation competent vectors are essentially live virus vaccines and share some of their advantages and disadvantages. They should be inexpensive to produce, and amplification in the vaccinee should allow immunization at a relatively low dose. The biological characteristics of the parent virus backbone should contribute to the characteristics of the vector: for example, the capability for oral immunization with poliovirus-based vectors. However, the ability of propagation competent vectors to amplify in the host also imposes several challenges. The first is the necessity of maintaining an attenuated phenotype. In most of these systems, the insertion of the exogenous gene is itself a strongly attenuating mutation (however, see Ref. 25), and this can be supplemented with other defined attenuating mutations to make reversion a rare event. Other issues include stability of the inserted gene, limits on inserted gene size, and induction of strong vector-specific immunity. Induction of strong vector-specific immunity may limit the ability to boost or to be used subsequently in the same individual for another indication. In those instances where an existing commonly administered human vaccine or common human pathogen is utilized as the vector, the ability to vaccinate with the exogenous gene in the first place may be severely limited by preexisting immunity to the vector itself.

Replicon Vectors Replicon vectors derived from positive strand RNA viruses are essentially self-replicating machines designed to amplify the mRNA for the exogenous genes that they carry. By substituting an immunizing gene for one or more viral structural protein genes, the immunizing gene can be expressed to high level. The replicon genome can be packaged into virions by supplying the structural proteins in trans, thus producing a replicon particle for specific delivery of the replicon genome in vivo in the form of a suicide vector incapable of propagation beyond the initially infected cell. Alphaviruses The antecedents of alphavirus replicon systems are found in the work of the Rice, Schlesinger, and Strauss laboratories who constructed the first cDNA clones of alphaviruses, mapped the cis-acting signals required for alphavirus RNA replication and encapsidation, and showed that alphavirus RNAs could be modified to express exogenous genes (5–7,26–31). Replicon expression systems have now been derived from several alphaviruses including SFV (32), SIN (33), VEE (34,35), and S.A.AR86 (36). A replicon system based on a related Togavirus, rubella has also been described (37). The structural protein genes, normally expressed to high level from a subgenomic mRNA, are deleted from alphavirus

Chapter 35: RNA Virus Replicon Vaccines Table 1 List of RNA Virus Vectors Positive strand RNA virus vectors

Negative strand RNA virus vectors

Propagation-competent Alphavirus Sindbis virus, VEE virus, Semliki forest virus Coronavirus TGEV, mouse hepatitis virus, SARS coronavirus Picornavirus Poliovirus

Propagation-competent Rhabdovirus VSV, RV

Flavivirus Yellow fever virus Chimeric virus Flavivirus Yellow fever virus, dengue fever virus Nonpropagating replicons Alphavirus Sindbis virus, Semliki forest virus, VEE Coronavirus TGEV Togavirus Rubella virus Picornavirus Poliovirus, rhinovirus, mengo virus Flavivirus Kunjin virus

Paramyxovirus Human or bovine parainfluenza virus 3 Human RSV Sendai virus, Newcastle disease virus Measles virus, simian virus 5 Influenza virus Chimeric virus Paramyxovirus Bovine RSV, human or bovine parainfluenza virus 3 Nonpropagating replicons Rhabdovirus VSV, RV Influenza virus

Abbreviations: RV, rabies virus; RSV, respiratory syncytial virus; TGEV, transmissible gastroenteritis virus; VEE, Venezuelan equine encephalitis; VSV, vesicular stomatitis virus.

replicon systems and replaced by the immunizing gene. Thus, the gene of interest is expressed in replicon-infected cells to levels approaching 20% of the total cell protein. In the SFV and SIN systems, high level expression depends on the presence of a translational enhancer sequence extending from the subgenomic mRNA start site approximately 200 nucleotides into the capsid gene (38,39). Although the cells are eventually killed as a result of replicon infection, the absence of the structural protein genes prolongs the period of maximal expression (40). Moreover, mutant alphavirus replicons have been selected, which are capable of persistent infection and expression (41–44). Packaging of alphavirus replicon genomes into replicon particles has been accomplished either by co-electroporation of replicon RNA and helper structural protein genes transcribed in vitro (32–35) or by the establishment of stable cell lines constitutively expressing transcripts for the structural proteins (45). In the packaging cell lines, the helper sequences are under the control of the alphavirus subgenomic RNA promoter, so that although they are constitutively transcribed from the cellular DNA as part of a larger mRNA, they are not transcribed from the subgenomic promoter or translated until introduction of the replicon RNA, either by electroporation or by infection with previously packaged replicon particles. Complementation occurs between the replicase functions encoded by the replicon RNA and the structural proteins supplied by the helper RNAs. Replicon particles are assembled that contain

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only the replicon RNA due to the absence of a cis-acting packaging signal in the helper RNAs. Alphavirus RNAs are capable of low-level recombination (46), and alphavirus particles can copackage multiple RNAs (33). In the context of alphavirus packaging systems, either can result in the production of propagation competent genomes. Expressing the capsid and glycoprotein genes from separate helper RNAs significantly reduces the generation of propagation competent virions contaminating replicon particle preparations (35,45,47). Replicon particles provide an efficient system for delivery of the replicon genome into cells in vivo. The effectiveness of the VEE replicon system may be attributable in part to the ability of VEE replicon particles to target and replicate within dendritic cells in lymph nodes of mice (48) and primates (West et al., unpublished data). Human monocyte-derived dendritic cells (DCs) also are targets for VEE replicon particles ex vivo, in which they induce maturation and active presentation of antigen to T cells (49,50). SIN variants, selected from laboratoryadapted strains for increased ability to target dendritic cells, may improve the ability of SIN replicon particles to induce immune responses (51). However, other studies suggest that wild-type SIN itself targets efficiently to dendritic cells in vivo (52). SFV replicon particles also appear to target lymphoid tissue (53), but do not infect professional antigen-presenting cells (54). Perri et al. (55) utilized a chimeric alphavirus system, combining SIN glycoproteins for targeting to cells in vivo and the VEE replicon genome to reduce sensitivity to interferon for better expression. Replicon RNAs derived from alphaviruses also have been delivered into animals directly (56–60), although degradation of the RNA prior to entry into cells may limit this approach. Alternatively, cDNAs driven by eukaryotic promoters have been used to express self-replicating replicon RNAs (61–66). In the case of cDNA delivery, the efficiency of transcription in and exit from the nucleus may limit effectiveness in a vaccine context (67). Alphavirus replicon particle vaccines have been tested in a variety of animal models of disease. Humoral, cellular and mucosal immunity have been demonstrated in mice, and in primates there is clear induction of both antibodies and cytotoxic T cells. Induction of protective levels of immunity and/or protection against challenge has been demonstrated with VEE replicon particle vaccines in rodent models of laboratoryadapted influenza (35); a human pathogenic Hong Kong origin H5N1 virus (68); Lassa fever, Ebola, Marburg, Anthrax, and botulinum toxin (69–74); staph enterotoxin (75); Norwalk virus (76,77); Lyme disease (78); gonococcus (79,80); SARS (81); orthopoxviruses (82); dengue fever (83); cytomegalovirus (84,85); respiratory syncytial virus (RSV) (86,87); and human metapneumovirus (88). Protection with VEE replicon particles also has been reported for equine arteritis virus in horses (89,90). In primates, partial protection against simian immunodeficiency virus (SIV) challenge has been demonstrated (91–94), and complete protection of primates against a high dose challenge of Marburg virus was achieved (95). Immunization with VEE replicon particles expressing the E7 protein from human papilloma virus 16 completely protected mice against tumor establishment in a murine model and led to eradication of established tumors in 67% of the animals (96,97). Homologous dendritic cells infected with VEE replicon particles (VRP) expressing her2/neu were effective in both prophylactic and therapeutic settings in wild-type mice but not in transgenic mice harboring

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the rat neu gene (98), although see also Wang et al. (99) and Nelson et al. (100). SIN-, SFV-, and SFV/VEE chimera-based replicon vectors, either as naked RNA, as DNA launched replicon genomes or replicon particles, also have induced varying degrees of immune response and protection in animal challenge models. These include influenza in mice (60,101–104 ), infectious bronchitis and infectious bursal disease viruses in chickens (105,106), flaviviruses in mice and sheep (60,107,108), herpes simplex in mice (64), tumor immunotherapy (109–116), immunodeficiency virus models in rodents and macaques (56,117–131), hepatitis C in conventional and HLA-A2.1 transgenic mice (132–134), Plasmodium falciparum in mice (59,103), RSV in mice (60,135), classical swine fever virus (136) and swine vesicular disease virus (137) in pigs, parainfluenza virus type 3 in mice (138), melanoma antigens (139,140), HPV (141–143), measles (144,145), tuberculosis (146), Brucella (147) and Chlamydia (148). Both a systemic and mucosal adjuvant effect is observed with alphavirus replicon particles even when inoculation is from a nonmucosal site (149). Therefore, these, and most likely replicon particles from other virus groups, serve two functions as vaccines: they produce the immunogen and they provide a significant adjuvant activity through induction of the innate immune response (149,150;151). In the case of alphavirus replicon particles, this represents a dichotomy of sorts in that these two activities may influence the immune response in opposing directions with a high level innate response depressing the expression of the vectored immunogen. Therefore, optimization of the adjuvant function (in the absence of transgene expression) and supply of immunogen by other means may ultimately constitute the most efficient vaccine application of these vectors. Picornaviruses Kaplan and Racaniello (152) identified sequences not essential for replication of poliovirus RNA, setting the stage for Ansardi et al. (153), who devised a poliovirus replicon system in which immunizing genes replace the P1 segment of the polio genome. Transfection of the replicon RNA and co-infection with vaccinia expressing the substituted polio structural protein genes results in the production of poliovirus replicon particles as well as the vaccinia helper. The polio replicon particles may be amplified by continued passage of the mixed population followed by separation of the much larger vaccinia virions from the polio replicon particles. This system has been utilized for immunization against a variety of bacterial and viral pathogens [e.g., Helicobacter pylori (154,155), HIV/SIV (156–159), tetanus toxin C-fragment (160), HPV (161)] and for tumor immunotherapy and viral induced lysis of tumors (162,163). Following from the poliovirus example, other picornaviruses, such as rhinoviruses (164) and mengo virus (165), also are being developed as vaccine vectors. Flaviviruses Flavivirus genomes are organized as a single long ORF encoding the structural proteins at the 50 end and the replicative nonstructural proteins in the 30 portion of the genomic RNA. Replicons of Kunjin virus have been constructed in which the structural proteins are replaced with the gene of interest in a cDNA clone of the virus genome (166,167). Much like the poliovirus replicons, electroporation of in vitro transcripts from the modified cDNA results in synthesis of the heterologous gene. Packaging of the Kunjin replicon genome into

flavivirus-like particles is accomplished by supplying the structural proteins in trans from an SFV replicon (168). Whether delivered into an animal as a naked RNA, a cDNA placed behind a eukaryotic promoter, or as a Kunjin replicon particle, this system induces humoral and cellular immune responses to heterologous antigens (169). A noncytopathic version of the Kunjin replicon has also been developed and may improve the capacity for immunization and/or enable gene therapy applications (170). Coronaviruses Coronavirus replicons are in the early stages of development but hold promise as vaccine vectors because of their potential for expression of large gene cassettes. Heterologous genes have been inserted into the genome of transmissible gastroenteritis virus after deletion of genes required for either virus replication or assembly. The required genes are supplied in trans by a helper virus (171) or by an alphavirus replicon expressing the helper genes (172). A potential problem, high level recombination, has been solved by incorporating synthetic cis-acting intergenic sequences, which produce nonviable recombinants with native coronaviruses (173). Advantages and Disadvantages of Replicon Vectors Replicon vectors combine some of the features of live virus and subunit vaccines. They initiate a partial replication cycle in vivo that allows production of immunizing genes in the context of viral replication, much as would occur with a live virus, including the induction of an innate response. The major product of that replication, however, is one or more protein subunits derived from the target pathogen. In a sense, replicon vectors represent the implantation of a subunit vaccine factory into the vaccinee, using the vaccinee’s own cells for in situ production of the subunit vaccine. The absence of viral structural protein genes in the replicon RNA allows immunization with the vectored immunogen without raising high levels of immunity to the replicon particles themselves, facilitating booster inoculations as well as sequential immunization of the same individual with the same vector expressing genes from other pathogens. Positive strand RNA replicon vaccines can be delivered as self-replicating naked RNAs or as cDNAs from which self-replicating RNA replicons can be transcribed by cellular polymerases. This feature may facilitate the rapid development of new vaccines, as Good Manufacturing Practice (GMP) production of DNAs is straightforward and DNA vaccines will have been well characterized. The deletion of the vector’s structural protein genes has two implications. First, it leaves room for larger inserted sequences than the related propagation competent vectors, and second, it makes replicon vectors inherently safe. Additional safety features can be built into the system, for example, using helper systems that reduce or eliminate the possibility of regenerating a propagation competent virus, including known attenuating mutations in the replicon genome and/or the structural protein genes used for packaging into replicon particles, and developing sensitive assays for the detection of propagation competent viruses in replicon particle preparations. However, a number of practical questions remain. Although several phase I human clinical trials have been conducted or are in progress, no results of these trials are yet available in the peer-reviewed literature for any of these systems, and for some, primate experiments have yet to be initiated. GMP production of replicon particles may be problematic, with low relative yields in approved cell

Chapter 35: RNA Virus Replicon Vaccines

substrates and no packaging cell lines yet established in cell substrates likely to be approved for GMP vaccine production. These difficulties will be magnified in adapting these processes to commercial scale. Notwithstanding these issues, replicon vaccines derived from positive strand RNA viruses have shown tremendous promise in experimental systems and certainly merit continued effort to resolve these potential limitations for progression into the clinic. A number of review articles have been published on expression systems derived from positive strand RNA viruses (92,167,174–192).

VACCINE VECTORS DERIVED FROM NEGATIVE STRAND RNA VIRUSES Negative strand RNA viruses share advantages with positive strand viruses when configured as vaccine vectors. Both have evolved mechanisms for high-level protein expression, and neither directs the integration of foreign genetic material into the genome of the host. However, now that systems have been established for the efficient recovery of several negative strand RNA viruses from molecular clones and specific signals for gene expression have been defined, members of this group of viruses offer additional advantages. First, viruses with segmented genomes and/or filamentous nucleocapsids can more easily accommodate additional whole gene segments, or large gene insertions. Second, many are infectious by the intranasal (IN) route and vectors derived from them can deliver heterologous immunogens to the respiratory mucosa. Third, multiple serotypes exist in many cases, which facilitate effective booster strategies. Finally, in several cases, safe and effective vaccine strains have been reproduced as full-length cDNA clones and are available as well-documented starting points for development of vaccine vectors. The biology of negative strand RNA viruses and their generation from cDNA clones has been extensively reviewed in Neumann et al. (193), and will be mentioned only briefly here.

Nonsegmented Negative Strand RNA Viruses Several laboratories have developed and used reverse genetic systems to study the unique modular genome organization of this group of RNA viruses. These combined discoveries have enabled the use of several nonsegmented negative strand RNA viruses as vaccine vectors, and have launched a distinct field of study, extensively reviewed by Bukreyev et al. (194). The main points will be highlighted here. Rhabdoviruses The best-known members of the Rhabdovirus family are vesicular stomatitis virus (VSV) and RV, both of which have been exploited as vaccine vectors. Their genomic RNA is approximately 11 kb in length and encodes (in order from 30 –50 ) nucleocapsid protein (N), phosphoprotein (P, previously NS, a cofactor in viral RNA synthesis), matrix protein (M), spike glycoprotein (G), and RNA-dependent RNA polymerase (L), all of which are found in the virus particle. The level of gene expression is directly related to gene position relative to the 30 end of the RNP template, and rearrangement of the viral genes leads to slower growth in cultured cells and attenuation of virus virulence in the animal host (195). These rearranged genomes are stable on passage, as homologous recombination

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does not occur. There are three known serotypes of VSV, but only a single RV serotype. Specific mutations in both the VSV and RV glycoproteins have been shown to attenuate virulence (see below), and live attenuated vaccines for rabies are being used for oral immunization of wildlife (196). VSV has a wide host range, from insects to man, and causes a mild, flu-like illness in humans. Laboratory animals can be infected by VSV using subcutaneous, intracranial, or IN routes. Schnell et al., working with RV, first demonstrated cDNA-based replication of a full-length recombinant rhabdovirus genome by using transfection of T7-driven RNA expression plasmids for the transcription of the complete genome complement (or antigenome) and N, P, and L mRNAs, followed by infection with vaccinia virus expressing T7 bacteriophage RNA polymerase (4). This strategy has been used successfully to produce recombinant VSV (197) and several paramyxoviruses (see below), and has been refined by replacing vaccinia virus with a T7-polymerase-expressing baby hamster kidney (BHK) cell line (198,199) or a cotransfected T7- polymerase expression plasmid (200,201). The demonstration of stable reporter protein expression from a sixth VSV gene, flanked by minimal start and stop signals and inserted between the G and L genes, followed soon after (202) (Fig. 1A.1). This basic strategy has been used in construction of numerous expression and vaccine vectors (203, also reviewed in Refs. 194,204–206), including those with two genes inserted into two different intragenic sites (207), or cocktails of two different vaccine vectors (208). Advantage has been taken of the signature 30 to 50 attenuation of transcription to modulate the level of gene expression. This was first shown with endogenous virus genes (209) and later with inserted heterologous genes (210,211). As predicted, the insertion of genes into more 30 proximal intergenic sites leads to higher-level expression and a stronger immune response. The ability of VSV to incorporate heterologous glycoproteins into its virions (212) suggested that heterologous glycoproteins expressed from VSV vectors would become part of analogous mosaic virions or pseudotypes. Viable viruses carrying additional genes for CD4, measles virus hemagglutinin (MH), or measles virus fusion protein (MF) contained varying amounts of the foreign protein inserted into their virion envelopes (203). The additional gene increased the length of the bullet-shaped particles, presumably due to increased length of the filamentous ribonucleocapsid, and reduced, to varying degrees, the level of virus replication in cultured cells. In many cases, but not all, no VSV-specific sequence was needed to drive incorporation of the additional glycoprotein into the virion envelope. The efficiency of VSV G incorporation into virions was not affected, suggesting that the new membrane protein occupied extra space in the envelope. By electron microscopy, each virus particle contained both proteins. This type of replication-competent VSV vaccine vector has been tested in animal models against measles virus (MV) (213), influenza virus (214,215), RSV (216,217), HIV-1 (208) bovine viral diarrhea virus (218), filoviruses (219,220), Lassa fever virus (221), SARS virus (222), and papillomavirus (210). Although the VSV vaccine vector expressing measles H protein displayed H protein on its surface, it nevertheless was able to induce protective levels of neutralizing antibody in the presence of maternal antibody, in contrast to the attenuated live virus measles vaccine (213). This result demonstrates the power of the recombinant vaccine vector approach in cases where vaccination in the presence of maternal antibody is needed to prevent dangerous early childhood infections.

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Two areas of concern surround the use of propagation competent VSV vectors: (i) the potential for serious veterinary disease, the febrile, flu-like illness associated with human infection, and the residual reactogenicity in mice of IN-administered vaccine vectors and (ii) induction of levels of anti-vector antibody that prevent effective booster immunizations (215,223,224). Attenuating mutations have been identified and tested in mice (215,225). In addition, effective boosting in the presence of anti-VSV antibody has been achieved by constructing vaccine vectors with G proteins of three different serotypes (224), and by using a heterologous prime-boost approach with VSV- and RV-derived vectors (226). In an alternate approach, both vector virulence and antivector immunity are addressed by the use of G-deleted, single cycle vectors that carry a heterologous glycoprotein gene and can be maintained only on cells that provide G in trans (Fig. 1B). Using these ‘‘packaging’’ cells, nonpropagating virions are produced. Such vectors were constructed expressing influenza HA, RSV G, or RSV F, and induced protective immune responses in mice without pathogenicity (217,225). However, when the G-deleted vector expressed an HIV-1 gp160 chimeric protein with the VSV G transmembrane and cytoplasmic domains, an infectious surrogate virus was produced, which grew efficiently in HIV-susceptible cells (227) (Fig. 1). This was also true for G-deleted vectors carrying the glycoprotein gene for Marburg, Ebola, or Lassa fever viruses (219) and, in reverse, for vectors expressing cellular CD4 and CXCR4, which mediated entry of vector particles into HIV-infected cells with gp160 on their surface (228). The utility of this approach may depend, therefore, on the ability of the heterologous glycoprotein to produce a propagation-competent chimeric virus, and its resulting biological properties. Direct comparison of immune responses in mice induced by inoculation of nonpropagating and replication-competent vectors showed the single cycle vector to be less immunogenic by the IN route (225), but essentially equivalent when inoculated intramuscularly (204,229,230). Whether or not these G-deleted vectors induce anti-vector antibody in mice also appears to depend on the route of inoculation (225,229). Advantages of the vector system based on RV include the availability of an attenuated vaccine strain shown to be safe in many animal species and the possibility of oral immunization (231). RV-based vectors expressing HIV, SIV, and SARS-CoV genes induced humoral and cellular immune responses in mice (232,233) and macaques (234). A single-cycle, P-deleted RV has been engineered and shown to be safe and immunogenic in mice; however, its use as a vaccine vector has not been explored (235). As shown for VSV, G-deficient RV vectors expressing a chimeric HIV-1 gp160-G protein (with the RV G protein cytoplasmic domain) contained gp160 in their envelope, showed reduced replication in vitro compared to G-containing vectors, and displayed the cell tropism of HIV-1 (236). Paramyxoviruses The Paramyxovirus family includes two subfamilies, the Paramyxovirinae subfamily, with the respiroviruses (e.g., Sendai virus, human parainfluenza, and bovine parainfluenza), the rubulaviruses [Newcastle disease virus, simian virus 5 (SV5)] and the morbilliviruses (measles and rinderpest viruses), and the Pneumovirinae subfamily, with the pneumoviruses (human and bovine RSVs) and the metapneumoviruses. Paramyxoviruses contain nonsegmented negative-sense RNA genomes in filamentous ribonucleocapsids enclosed in polymorphic envelopes.

They share patterns of gene expression and regulation with the simpler rhabdoviruses but carry additional genes. For example, all paramyxoviruses have two distinct glycoprotein spikes, one for attachment and the other for fusion with the host cell membrane. Reverse genetics systems using techniques analogous to those described for rhabdoviruses have been reported for MV, RSV, Sendai virus, Rinderpest virus, human parainfluenza type 3 (hPIV-3), and SV5, (reviewed in Ref. 237), Newcastle disease virus (238,239), bovine PIV-3 (240), and human PIV1 (241). The construction and testing of several vaccine vectors based on the paramyxoviruses is described in detail by Bukreyev et al. (194). An important part of this effort has been the identification and characterization of attenuating mutations, either point mutations used in combination or deletions of genes that are not required for replication in vitro, but which act to increase virulence in vivo. Using these attenuated genomic backgrounds, additional genes expressing foreign immunogens have been inserted to produce bivalent vaccines (194,242–245). A second strategy is the construction of antigenic chimeric viruses, analogous to the surrogate viruses described above for VSV in which the vector glycoprotein genes are replaced by those of another virus. Examples are the replacement of the G and F genes of recombinant bovine RSV with human RSV G and F (246) and the expression of HPIV1 HN and F glycoproteins in the HPIV3 genomic backbone (247). Safety was increased by using an attenuated virus (248) or a host range restricted virus, such as bovine RSV or PIV3, as the carrier (240,246). Although these antigenic chimeras are not designed to be bivalent vaccines, in some cases, the cellular immune response to proteins expressed from the remaining vector genes can be partially protective (249). The next step was the insertion of additional genes into the antigenic chimeric virus vectors to produce true bivalent vaccines, such as substitution of HPIV3 and human RSV glycoproteins or HPIV3 and hMPV glycoproteins into the bovine PIV3 backbone (250,251). The reverse genetics approach for MV has led to rescue of recombinant MV expressing several heterologous reporter proteins and immunogens (recently reviewed in Ref. 252). This work is based on a full-length cDNA of the Edmonston B vaccine strain, which has a long history of safe and effective human use. Studies are proposed to determine whether MVbased vaccine vectors can be used as bivalent vaccines in infants and as vaccine vectors in adults already immunized against MV. SV5 is a nonpathogenic paramyxovirus that can infect humans but is not associated with any known disease. Recombinant SV5 expressing a foreign antigen induced high avidity antigen-specific cytotoxic T lymphocytes in mice following a single IN inoculation (253). The biological properties of this vaccine vector warrant its further consideration for human use against respiratory pathogens.

Segmented Negative Strand RNA Viruses Influenza Virus Influenza virus is a member of the orthomyxovirus family, with a genome of eight segments of negative-sense RNA. The virus transmembrane glycoproteins, hemagglutinin (HA), and neuraminidase (NA) function together to access, attach to, and enter appropriate target cells, and the anti-influenza neutralizing antibody response is directed toward these proteins. Fifteen

Chapter 35: RNA Virus Replicon Vaccines

distinct HA subtypes and nine different NA subtypes have been identified, which can be alternated in sequential immunizations for improved boosting (see sect. ‘‘Rhabdoviruses’’). Several reverse genetics systems have been described for influenza virus, reviewed by Neumann and Kawaoka (254) and Neumann et al. (193), and improvements continue to be made (255). These have been used in various strategies to express foreign genes from influenza virus vectors (reviewed in Ref. 256, 257). One approach has been the expression of a foreign peptide as part of a chimeric HA or NA protein (256,258–268). Although these vectors have induced specific humoral and cellular immune responses against several disease agents, this strategy is limited with respect to the amount and character of the foreign sequence that can be inserted without detrimental effects on the viability of the recombinant vector. Foreign sequences have also been inserted into the NS1 gene, a nonstructural protein that may have fewer size constraints than a structural protein, is abundant in influenzainfected cells, and immunogenic in animals (269,270). Also, the abrogation of its function as an interferon antagonist has been shown to significantly attenuate influenza virus virulence (271– 273). A recombinant NS genome segment was engineered to express a truncated NS1 protein fused to a self-cleaving footand-mouth disease virus (FMDV) protease domain followed by an HIV-1 Nef-derived polypeptide. This segment was rescued into viable virus using a high-efficiency helper system for NS mutants (274). This virus vector was attenuated in mice and induced a significant Nef-specific immune response. Strategies for expression of entire foreign genes include engineering of influenza virus genome segments to express bicistronic mRNAs separated by an internal ribosome entry site (263,275) and expression of a full-length foreign gene from an additional influenza virus gene segment (276). Vaccinia-T7-based (277) and plasmid-based (278) systems have been established for the production of nonpropagating influenza virus-like particles (VLPs). An RNA polymerase Idriven plasmid was engineered to express a vRNA with an antisense copy of green fluorescent protein. VLPs containing the synthetic vRNA were packaged by cotransfection of plasmids expressing the influenza structural proteins. By analogy to replicon particles of positive-strand viruses, single-cycle vector particles that contain a vRNA expressing a gene of interest as well as vRNA segments for PA, PB1, PB2, and NP (required for transcription and replication of the foreign sequence) would deliver the foreign gene to influenza virus susceptible cells for in vivo expression, but be unable to spread in the host. Also, a nonpropagating influenza virus vaccine vector could be based on the work of Watanabe et al. (279), who engineered an NS2-knockout virus that is replication-defective and highly immunogenic in mice.

CONCLUSION RNA virus genomes, reconfigured to express heterologous antigen genes, have great potential as vaccine vectors. Levels of expression are generally high, the plasticity of RNA genomes will allow innovative use of their coding capacity, knowledge of virulence determinants will insure safety, and the large variety of potential vaccine vectors will obviate problems of anti-vector immunity. However, this field is relatively young, and significant hurdles must be surmounted as RNA virus vectors progress from the laboratory to human use vaccine products. Few of these concepts have moved from tests in mice

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to experiments in nonhuman primates, and fewer still have progressed to human trials. In addition, some of these laboratory-derived systems are not particularly well suited to GMP production at commercial scale. Nevertheless, the potential of RNA virus–based replicon strategies for improvement of existing vaccines and for derivation of new ones places these rapidly developing vaccine concepts at the leading edge among new vaccine technologies.

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36 Engineering of Attenuated Salmonella enterica Serovars for Use as Live Vector Vaccines James E. Galen, Marcela F. Pasetti, Sharon Tennant, Marcelo B. Sztein, and Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION Attenuated strains of Salmonella enterica serovar Typhi (S. Typhi) or S. enterica serovar Typhimurium (S. Typhimurium) bacteria have been utilized extensively over the years to create live vectors that deliver protective antigens from unrelated target pathogens to the immune system, eliciting immunity against both the bacterial carrier strain and the unrelated pathogens (1,2). Salmonella are particularly well suited for this purpose: they can be delivered orally; Salmonella target M cells overlying gut-associated lymphoid tissue (inductive sites for immune responses); they are readily internalized by dendritic cells and macrophages; and Salmonella elicit broad immune responses. The breadth of this immunity includes serum antibodies, secretory IgA intestinal antibodies, and an array of cellmediated immune responses (CMI), including specific cytotoxic lymphocytes and interferon-g (IFN-g)-secreting effector T cells (3). S. Typhimurium live vectors tested in murine animal models have been extraordinarily successful in stimulating broad humoral and cellular immune responses against a variety of foreign antigens from other bacteria, viruses, protozoa, and helminths, with challenge models documenting the ability of many of these S. Typhimurium vaccines to vaccinate mice against challenge with the heterologous pathogen (4–7). Several groups of investigators have reasoned that, based on successes achieved using S. Typhimurium–based live vectors, it should be relatively straightforward to adapt S. Typhi vaccine strains for use as live vectors in humans to achieve similar levels of safety and immunogenicity to foreign antigens. However, few constructs have advanced to phase I clinical trials, and immune responses have been disappointing compared with analogous studies with S. Typhimurium in mice. In this review, we discuss the various challenges that must be addressed to enable S. Typhi to function successfully as a live vector and summarize the impressive progress made in foreign antigen expression technology, highlighting the need for fresh clinical trials to test vastly improved candidate live vector vaccines.

SELECTION OF ATTENUATED SALMONELLA TYPHI STRAINS TO SERVE AS LIVE VECTORS In addition to live oral typhoid vaccine strain Ty21a (that was derived in the early 1970s by chemical mutagenesis), recombinant DNA technology has succeeded in creating several

attenuated S. Typhi strains that have been clinically well tolerated and immunogenic in eliciting anti-Typhi immune responses. Accordingly, these strains have been proposed to serve as single-dose live oral typhoid vaccines (as opposed to less immunogenic Ty21a, which requires three or four doses). Among the best known of these strains are CVD 908, CVD 908-htrA, CVD 909, Ty800, and M01ZH09 (8–12). These strains have also been proposed to serve as live vectors. However, there is increasing concern that a strain that may be ideal as a single-dose live oral typhoid vaccine may not necessarily function as an ideal live vector. Strains that are finely tuned to be safe and nonreactogenic live oral typhoid vaccines may not be hardy enough to serve optimally as live vectors because of the various stresses associated with the expression of foreign antigens. For example, within a series of progressively attenuated strains developed at the Center for Vaccine Development, the less attenuated CVD 908 may be best for use as a live vector, while further derivatives CVD 908-htrA and CVD 909 may be optimal as oral typhoid vaccines. To date, this concern has not been systematically examined, and attempts to minimize the metabolic impact of foreign antigen expression have focused on synthesis of the antigen itself and not on the basic fitness of the live vector.

INNOVATIONS IN EXPRESSION PLASMID TECHNOLOGY A wide variety of plasmid-based expression technologies are available to achieve production of sufficient levels of antigens to generate appropriate immune response(s). However, often overlooked in live vector engineering is the effect that expression plasmids (and the heterologous antigens they typically encode) can exert on the fitness of a live vector (13). The metabolic burden imposed by resident antigen expression plasmids has the potential to render a vaccine strain overattenuated and subimmunogenic. Although spontaneous plasmid loss would remove any metabolic burden and allow plasmidless bacteria to quickly outgrow the population of plasmid-bearing bacteria, such a shift in the live vector population would reduce its capacity to stimulate adequate immune responses to the foreign antigen. Most nontyphoidal Salmonella serovars associated with gastroenteritis in humans harbor a virulence plasmid. In contrast, S. Typhi, the agent of typhoid fever, does not. Moreover,

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prior to the late 1980s, S. Typhi did not typically carry R factors except in time-limited outbreaks in Mexico (1971–1973), Vietnam (1973–1975), and Peru (1980) (14,15). However, since 1989, S. Typhi strains carrying resistance plasmids (R factors) encoding resistance to multiple clinically relevant antibiotics have been commonplace in Asia. These resistance plasmids are predominantly members of a family of self-transmissible factors of the IncH incompatibility group that transfer themselves by conjugation using plasmid-encoded sex pili of the H complex (16). Plasmids within the IncH group are further classified into two subgroups, IncHI and IncHII, on the basis of the permissive temperature at which plasmids can be transferred at high efficiency. IncHI factors are transferred with high efficiency at low temperatures between 268C and 308C, while optimum mating transfer for members of the IncHII group is not thermosensitive and occurs efficiently at 378C. IncHI plasmids can be further divided into subgroups 1, 2, and 3, on the basis of differences in DNA homology and compatibility with the classic Escherichia coli F factor (16). Contrary to many other enteric pathogens, R factors associated with S. Typhi belong almost exclusively to the IncHI1 subgroup (14,15,17–20). The unique predominance of IncHI1 plasmids found naturally among clinical isolates of S. Typhi suggests that stable maintenance of unrelated expression plasmids introduced into such strains may not be straightforward. Therefore, the further development of attenuated and clinically acceptable S. Typhi vaccine strains into live vectors carrying engineered plasmids expressing foreign antigens becomes problematic. Reengineering of IncHI1 replicons for use as expression plasmids, although theoretically feasible, has, to date, not been attempted. In addition, the copy number of these plasmids is one to two copies per chromosomal equivalent and, therefore, provides little advantage over chromosomal integration systems for expressing heterologous antigens. However, closer inspection of the general organization of naturally occurring plasmids found in clinical isolates of S. Typhi (as well as those found in unrelated environmental strains of bacteria) hints at the possibility of designing a stable multicopy expression plasmid that could be used successfully with S. Typhi attenuated vaccine strains to construct polyvalent live vector vaccines. Paradigms of plasmid organization that promote stable plasmid replication and maintenance have been described by Thomas (21,22) to include (i) a self-regulating origin of replication, (ii) an active partitioning mechanism to promote the nonrandom distribution of plasmids into bacterial daughter cells, (iii) a post-segregational killing system to remove plasmidless daughter cells from a growing population of bacteria, (iv) a multimer resolution mechanism for decatenating recombined plasmids that must segregate independently for proper inheritance, (v) a conjugative transfer system for plasmid mobilization between bacteria, and (vi) at least one locus that confers a selective advantage to the host bacterium. Redundancy is another recurring theme for naturally occurring plasmids, with two or more replication origins and partition functions often being present. Because of regulatory considerations, live vectors intended for human use cannot carry self-transmissible plasmids, and the use of selectable genes encoding resistance to ‘‘clinically irrelevant’’ antibiotics is currently being strongly discouraged by regulatory agencies. Development of expression plasmids for use in S. Typhi (as well as other) live vectors has, therefore, taken advantage of small nontransmissible multicopy replicons, which enable more DNA sequence to be

devoted to expression of heterologous antigens while minimizing the size and genetic instability of the replicons. As briefly summarized below, efforts to optimize stable and immunogenic plasmid-based expression systems for these foreign antigens have been focused in several areas including (i) regulated expression of foreign antigens, (ii) reduction of antigen toxicity, (iii) ‘‘engineering’’ immune responses by targeted antigen expression, (iv) genetic optimization of plasmid stability, and (v) the recent development of nonantibiotic plasmid selection systems.

Improvement of Foreign Antigen Expression Regulated expression of multicopy foreign antigen cassettes reduces the overall metabolic burden imposed by multicopy expression plasmids, thereby improving the fitness and immunogenicity of a vaccine construct. Such controlled expression can be achieved at the level of transcription, translation, or both. Chatfield et al. (23) demonstrated that constitutive expression in S. Typhimurium, using the powerful tac promoter [Ptac] to express tetanus toxin fragment C (frag C) from a multicopy plasmid, resulted in undetectable frag C–specific antibodies after oral immunization of mice. Isogenic expression using the reengineered nirB promoter Pnir15, which responds to the environmental signal of low oxygen, elicited antitoxin antibodies that protected orally immunized mice against lethal tetanus toxin challenge. It was further shown that plasmids carrying the constitutive Ptac–frag C cassette were rapidly lost in vivo from bacteria colonizing deep tissues (23). This concept of using ‘‘in vivo induced’’ expression cassettes to enhance the stability of multicopy plasmids was soon expanded to include eukaryotic antigens from parasites such as Schistosoma (24) and Leishmania (25). Subsequently, Bumann (26) clearly demonstrated that regulated expression of foreign antigens strongly influences the immunogenicity of live vectors by affecting colonization levels within the host. When comparing constitutive expression of green fluorescent protein (GFP) fusions from P tac versus regulated expression from the PhoQ/PhoPcontrolled PpagC promoter, Bumann showed that while both constructs induced comparable cellular immune responses, ~1000-fold lower doses were needed if the antigen was expressed from PpagC (26). A remarkable variation on the theme of enhancing plasmid stability through appropriate regulation of plasmid-borne genes has recently been described by Doyle et al. (27), who identified a gene encoding an H-NS-like repressor (sfh) that occurs naturally on self-transmissible IncHI1 factors found in S. enterica serotypes Typhimurium (16,17), Typhi (18), and Paratyphi A (28), as well as in Shigella flexneri 2a (27). H-NS is normally synthesized at constant levels within bacteria and regulates various chromosomally encoded genes through binding to AþT-rich regions, frequently associated with promoter regions (29–32). Doyle’s experiments demonstrated that small multicopy plasmids introduced into bacterial hosts can titrate the cellular pool of H-NS, resulting in less regulation of chromosomal genes and severe loss of fitness (27). Naturally occurring self-transmissible R factors (which often encode hundreds of heterologous proteins) have apparently solved this problem by encoding their own H-NS-like repressor to silence plasmid genes and thereby minimize the impact of incoming large plasmids on recipient metabolism. Inclusion of an sfh allele into S. Typhi expression plasmids poses an intriguing (and as yet untested) possibility for reducing the

Chapter 36: Engineering of Attenuated S. enterica Live Vectors

metabolic burden incurred by plasmid-bearing strains both at the intrinsic level of the plasmid DNA sequence and at the level of foreign gene expression.

Reduction of Foreign Antigen Toxicity It is not enough to optimize in vivo expression levels to secure an appropriate immune response (13). Problems with inherent antigen toxicity may diminish the colonizing ability of live vectors and lower the levels of antigen delivered to immunological inductive sites. In addition, proper folding may be required for conformationally specific epitopes to trigger protective serum antibody responses. For example, neutralizing serum antibodies against the critical 19-kDa carboxyl terminal domain of merozoite surface protein 1 (MSP-1) from Plasmodium falciparum are only observed when the six disulfide bridges of the terminal domain are properly folded (33,34); this is unlikely to occur efficiently for antigens synthesized within the reducing environment of the live vector cytoplasm. In attempts to address potential toxicity and protein folding problems, various antigen export technologies have been developed for surface expression or for the extracellular secretion of foreign proteins. One successful approach utilizes a novel surface display technology based on engineering of expression cassettes derived from the Pseudomonas syringe ice nucleation protein (INP); the versatility and promise of this strategy are illustrated by reports of the display of properly folded eukaryotic antigens such as the human immunodeficiency virus type 1 glycoprotein (gp)120 on the surface of E. coli (35), and construction of an immunogenic multivalent hepatitis B surface antigen/hepatitis C core protein displayed on the surface of the licensed S. Typhi vaccine strain Ty21a (36). In addition to surface display, secretion of heterologous antigens out of Salmonella live vectors has been reported by several groups to enhance the immune response to a foreign protein. Hess et al. (37) reported that cytoplasmic expression of the protective T-cell antigen listeriolysin O (LLO) within recombinant Salmonella vaccine strains did not confer protection in mice against lethal challenge with virulent Listeria monocytogenes. However, in-frame insertion of LLO within a truncated form of the E. coli hemolysin (HlyA) A allowed extracellular secretion of this fusion in the presence of the coexpressed E. coli HlyB/HlyD/ TolC export apparatus and resulted in protection against lethal challenge with L. monocytogenes. Similar results have been reported by other groups using type III secretion systems encoded either by Salmonella pathogenicity islands (SPI)-1 (38,39) or by SPI-2 (40) to elicit protection using secreted antigens from both prokaryotic and eukaryotic pathogens. A novel antigen export system has also been described, which is derived from a cryptic hemolysin encoded by cytolysin A (ClyA) within the chromosome of S. Typhi strains, including the clinically proven strains CVD 908-htrA (41) and Ty21a (42). The molecular biology of ClyA from S. Typhi is well characterized (42–45), and it has been conclusively demonstrated that unfused ClyA is exported out of bacteria via outer membrane vesicles (46). Such a mechanism for vesicle formation raises the intriguing possibility of engineering ClyA to export antigens out from live vectors that are otherwise potentially toxic when expressed cytoplasmically; these vesicles would also carry lipopolysaccharide (LPS), which might improve the immunogenicity of a foreign antigen. This ClyA-mediated export technology has been successfully applied to the development of S. Typhi–based vaccines

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carrying antigens from prokaryotic and eukaryotic organisms (47–49). The usefulness and versatility of this system has been extensively demonstrated using genetic fusions of ClyA to the cell-binding PA83 subunit of Bacillus anthracis anthrax toxin. Delivery of ClyA-PA83 protein fusions by the licensed typhoid vaccine strain Ty21a to mice (48) and by candidate vaccine strain CVD 908-htrA to mice and monkeys (49) was shown to elicit high titers of toxin-neutralizing antibodies in animals primed intranasally with live vector and boosted with the licensed BioThrax1 anthrax vaccine. These ClyA-PA83 protein fusions were demonstrated to be efficiently exported to the surface of attenuated S. Typhi strain CVD 908-htrA despite increasing the size of the ClyA export domain from its original 35 kDa to 118 kDa as a fusion protein (Fig. 1). The biological relevance of antitoxin responses against ClyA-PA83 was proven in work carried out with S. Typhimurium where mice were protected against a lethal aerosol anthrax spore challenge when oral immunization with live vectors expressed ClyA-PA83 but not when live vectors delivered PA83 fused to E. coli HlyA (5). It is, therefore, clear that the regulated expression of foreign

Figure 1 Plasmid-based expression of ClyA-PA83 on the surface of the Salmonella Typhi CVD 908-htrA live vector, detected by immunogold staining. Immunoelectron micrographs of (A) S. Typhi CVD 908-htrA expressing the ClyA-PA83 protein fusion, (B) S. Typhi CVD 908-htrA expressing unfused PA83 in the cytoplasm, and (C) S. Typhi CVD 908-htrA without any expression plasmid (negative control) incubated with mouse PA-specific antibodies and gold-labeled anti-mouse antibody. (D) S. Typhi CVD 908-htrA expressing the ClyA-PA83 fusion protein, incubated with negative serum and gold-labeled anti-mouse antibody. Bar: 0.25 mm. Abbreviations: ClyA, cytolysin A; PA83, Protective Antigen (83 kDa) from anthrax toxin.

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antigens in the appropriate bacterial compartment can have a profound impact on immunogenicity.

Engineering Immune Responses by Targeted Antigen Expression The type of immune response elicited by recombinant antigens expressed by live vectors largely depends both on the efficiency of antigen presentation and the capacity of the organism to target specific immune cells. Accessibility of sufficient amounts of antigen to the appropriate cellular compartment will directly influence the nature and strength of immune responses induced. Depending on whether humoral or cellular immune responses are required to provide protection against a given pathogen, targeting antigen delivery to the appropriate cellular compartment becomes critical. While surface-expressed antigens are known to preferentially stimulate humoral responses, refinements in antigen expression and delivery technology have allowed targeting other cellular compartments to enhance cellular immunity. Hly-mediated (via HlyA) antigen secretion into the phagosome was shown to enhance priming of CD4 and CD8 T-cell responses (37). A series of studies led by Russmann et al. showed that SPI-1 and SPI-2-mediated antigen delivery into the cytoplasm of antigen-presenting cells (APC) enhances T cell–mediated immunity (38,50,51). SPI-1-dependent translocation of Listeria peptides into the cytosol led to efficient major histocompatibility complex (MHC) I–restricted antigen presentation demonstrated by IFN-g production and cytotoxic responses by peptide-specific CD8 T cells, which conferred protection against lethal challenge with wild-type Listeria (38). Similarly, SPI-2-mediated antigen delivery resulted in efficient priming of central and effector memory CD8 T cells (51). Because of the efficiency in priming T cells, this live vectorbased strategy has been explored for prophylactic treatment of tumors (52).

Optimization of Plasmid Stability Further refinements to improving the immunogenicity of plasmid-based foreign antigens delivered by live vectors have addressed the inheritance of expression plasmids within dividing live vectors. To prevent plasmidless daughter cells from overtaking a growing population, conditionally lethal systems were engineered such that plasmid loss quickly led to cell death (53–55). One such system is based on the expression of the asd gene encoding aspartate b-semialdehyde dehydrogenase (Asd), an enzyme critical to synthesis of the cell wall and several amino acids (56). Loss of plasmids encoding Asd is lethal for any bacterium incapable of synthesizing Asd from the chromosome, resulting in lysis of the bacterium due to an inability to correctly assemble the peptidoglycan layer of the cell wall. The asd system thus improves the apparent stability of expression plasmids by removing plasmid-cured bacteria from the population (i.e., a post-segregational killing system). The asd system has been successfully employed in attenuated S. Typhimurium live vector strains (57) expressing a variety of antigens including tetanus toxin frag C (58), E. coli heat-labile enterotoxin (LT) (59), synthetic hepatitis B viral peptides, (60) and, more recently, Yersinia pestis F1 and LcrV antigens (61). Mice immunized mucosally with these recombinant strains elicited potent immune responses including serum IgG and secretory IgA. However, results were disappointing when the asd system was introduced into attenuated S. Typhi strains and tested in clinical trials. Volunteers immunized with

S. Typhi asd mutants expressing hepatitis B viral peptides from asd-stabilized plasmids failed to elicit responses to the foreign antigen (62). A variation of the conditionally lethal system to enhance plasmid retention involves expression plasmids that encode a self-contained toxin-antitoxin system in which the protective antitoxin is unstable and requires constant synthesis from resident expression plasmids; plasmid loss activates the toxin, again leading to cell lysis (63,64). To remove the random partitioning of multicopy plasmids during cell division, plasmid segregation functions were also introduced to ensure nonrandom inheritance of plasmids into all daughter cells (64). Quantitative in vitro analysis of plasmid retention clearly demonstrated that as toxin-antitoxin and partitioning maintenance functions were incrementally introduced, plasmid stability improved accordingly. Use of this plasmid maintenance system has recently progressed into preclinical trials in nonhuman primates, where expression plasmids have combined this maintenance system with the ClyA antigen export system to test the immunogenicity of ClyA-PA83 fusions. Monkeys primed mucosally (i.n.) with attenuated S. Typhi live vector CVD 908-htrA expressing ClyA-PA83 fusions were boosted three months later with a single parenteral dose of BioThrax1 vaccine. Notably, within seven days after administration of the single parenteral booster, robust toxin-neutralizing antibody levels were detected in serum (49). Another approach to improving plasmid stability that shows promise for improving the immunogenicity of foreign antigens borrows from motifs observed in nature that reduce the multimerization of plasmids. The cer-Xer recombination system of E. coli ColE1 replicons was the first site-specific multimer resolution system proven to decatenate plasmids and promote stability by increasing the number of functionally inheritable replicons (65,66). However, this locus depends on four chromosomally encoded host functions for multimer resolution (67), and the efficiency of this system will likely depend on the host background. Several analogously functioning but apparently self-contained resolution systems have since been identified in self-transmissible factors isolated from a variety of enteric strains (68,69). Using the crs-rsd site-specific resolution system, originally identified in the virulence plasmid pSDL2 from S. enterica serovar Dublin, Stephens et al. (70) observed that incorporation of this stability module into ColE1 replicons dramatically improved plasmid retention in S. Typhi vaccine strain CVD 908-htrA. Interestingly, the highest retention frequencies were observed only after additional transcription elements were incorporated into these expression plasmids to tightly regulate foreign antigen expression levels (70). This system awaits further immunogenicity testing in animal models.

Development of Nonantibiotic Plasmid Selection Systems As described above, one method for accomplishing both the selection and retention of expression plasmids, without the use of antibiotic selection, involves the construction of a conditionally lethal system. A clever variation of the balanced lethal nonantibiotic strategy for plasmid selection and maintenance involves construction of a conditionally lethal transcriptional control circuit in which the lacO-lacI operator-repressor genes controlling the E. coli lactose operon are engineered to control the synthesis of a chromosomally encoded protein critical for bacterial survival. Introduction of multicopy expression

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plasmids carrying lacO into the carrier organism titrates lacI repressor away from chromosomal lacO to enable synthesis of the required protein and bacterial growth. Loss of such an expression plasmid would then assure that surplus lacI binds to chromosomal lacO, shutting down synthesis of the essential protein and resulting in the death of plasmidless cells (71). The titration-repressor technology was incorporated into a low copy number plasmid encoding the F1 antigen from Y. pestis and transferred to S. Typhimurium. This vaccine construct protected five out of six orally immunized mice from subcutaneous lethal Y. pestis challenge. The stabilized expression plasmid was retained in vivo for two weeks (72). Another novel approach to nonantibiotic plasmid selection was recently reported by Fang et al. (73) and is based on immunity to the antimicrobial peptide microcin H47 (MccH47). Expression plasmids encoding the GFP test antigen and either the selection marker mchI, conferring immunity to MccH47, or the conventional bla gene, conferring resistance to ampicillin, were selected in both attenuated S. Typhi and Shigella flexneri 2a strains. Plasmids selected in Shigella flexneri 2a using MccH47 elicited GFPuv-specific humoral immunity comparable to that elicited by conventional plasmids. Interestingly, microcinselected plasmids elicited better GFP-specific IgG responses in S. Typhi live vectors than conventional plasmids. However, both plasmids proved much more unstable in S. Typhi than in Shigella flexneri 2a, emphasizing the challenge of stably maintaining expression plasmids in S. Typhi strains versus other enteric live vectors.

CHROMOSOMAL ANTIGEN EXPRESSION STRATEGIES The choice of using plasmids for expression of heterologous proteins is obviously not a simple matter of introducing multicopy plasmids into a live vector to achieve appropriate synthesis of the foreign protein of choice. In addition to copy number, maintenance functions to ensure plasmid stability must be considered, as well as the possible reengineering of the chromosome to create nonantibiotic plasmid selection systems. An alternate and perhaps simpler approach to heterologous antigen expression involves direct integration of expression cassettes into the chromosome. Following the deletion of a given chromosomal locus to create an attenuated vaccine strain, that locus can later be the site into which a cassette encoding the desired foreign antigen is placed. Chromosomal expression of foreign antigens eliminates the need for additional maintenance and selection systems. Theoretically, chromosomal expression also offers the possibility of easily constructing a multivalent vaccine (74), in which a large variety of chromosomal locations can be chosen for expression of distinct antigens, from either the same or several unrelated pathogens. However, as with plasmid-based systems, immunogenic levels of each foreign protein must be synthesized, and the balance between antigen synthesis and metabolic burden must still be assured. Not surprisingly, early attempts at chromosomal expression of heterologous antigens focused on the use of constitutive promoters to drive continuous synthesis of foreign protein in hopes of inducing the desired humoral and CMI responses (75,76). An early clinical trial generated some optimism and proof of principle for this general tactic (77). Of 10 volunteers who were immunized orally with two doses (eight days apart) of an S. Typhi–based vaccine expressing a truncated P. falciparum circumsporozoite protein (CSP) antigen under the control

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of the constitutive Ptac promoter, 2 of 10 volunteers developed significant rises in serum antibody to sporozoite antigens and a third subject developed CSP-specific CD8þ cytotoxic lymphocytes that lysed targets expressing CSP (77). This was the first demonstration in humans that S. Typhi live vectors could elicit cytotoxic lymphocyte responses to a foreign protein. Hohmann et al. (78) demonstrated in mice that foreign antigens expressed constitutively from the chromosome of S. Typhimurium failed to elicit antigen-specific immune responses. In contrast, expression of the identical antigen using PpagC, induced after phagocytosis of live vectors by murine macrophages, elicited strong serum IgG responses against the vectored antigen. It was hypothesized that the intracellular location in the APC and timing of heterologous antigen expression, rather than constitutive expression of large amounts of the protein, are critical in eliciting immunity to the foreign antigen. Building upon the observations of Hohmann, Stratford et al. (79) reasoned that the use of the more powerful PssaG in vivo inducible promoter to drive chromosomal expression within the phagosome would stimulate more robust antigenspecific immune responses. Since PssaG is a tightly regulated SPI-2 promoter that is induced 400-fold upon phagocytosis into macrophages (80), it was hypothesized that it might be feasible to construct a live vector in which foreign antigen expression is minimal prior to vaccination but becomes highest at critical immunity induction sites. Therefore, an expression cassette was assembled, encoding a PssaG-controlled eltB gene directing synthesis of the B subunit of LT from enterotoxigenic E. coli (ETEC). This cassette was then integrated to replace the deleted aroC gene of the S. Typhi vaccine candidate M01ZH09. Mice immunized i.n. with a single dose of live vector displayed potent antigen-specific IgG responses. When volunteers were immunized orally with two doses (56 days apart) containing either 108 or 109 colony-forming units (cfu) of the identical live vector constructs, 5 of 12 (42%) and 10 of 24 (42%) subjects, respectively, developed significant rises in serum IgG anti-LT following ingestion of the first low or high dose of live vector. Several additional subjects at each dosage level seroconverted ingestion of the second dose of vaccine so that in total 22 of the 36 subjects (61%) manifested a rise in serum IgG anti-LT (81); IgA antibody–secreting cell responses to LT were meager. Considering that LT B is a very strong antigen and also an adjuvant, the modest anti-LT responses recorded in the clinical trial were surprising. It is likely that a combination of further refinements in expression strategies, along with improved vaccination protocols, will be required to achieve potent immune responses in humans using Salmonella live vectors.

IMPROVEMENTS TO IMMUNIZATION STRATEGIES One emerging vaccination strategy that has proven effective at enhancing immune responses, particularly against poorly immunogenic antigens, is the ‘‘heterologous prime-boost’’ approach (82). The heterologous prime-boost strategy involves sequential administration of a target antigen, in different vaccine formulations, administered by the same or different routes. This strategy is capable of inducing both CMI and humoral responses that are superior to those achieved using a homologous prime-boost regimen with a given vaccine formulation. A variety of complex mechanisms are undoubtedly involved in controlling the magnitude and breadth of responses induced by prime-boost immunization. However, one central hypothesis

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holds that administration of the target antigen using two distinct delivery vehicles allows for expansion of antigenspecific memory T and B cells while minimizing anti-vector immunity that might hamper immunity to the foreign antigens (82,83). Upon antigenic reexposure, primed antigen-specific memory cells are believed to rapidly expand, mounting enhanced and broadened anamnestic responses. Initially, this approach was shown to improve immune responses against purified malarial and HIV antigens (84–88). More recently, this technique has been demonstrated to enhance the immunogenicity of live vector-expressed prokaryotic and eukaryotic heterologous antigens. Salmonella have been successfully used as priming agents, followed by subsequent boosting with the target antigen given as a purified protein in the presence of adjuvant or encoded by a DNA vaccine. Londono-Arcila et al. described for the first time the use of an S. Typhi–based live vector in a heterologous prime-boost regimen. In this study, attenuated vaccine strain CVD 908-htrA and its earlier parent strain CVD 908 were engineered to express Helicobacter pylori urease from plasmid-borne genes transcriptionally controlled by the stress-regulated promoter PhtrA (89). In mice primed i.n. with live vectors, followed by two subcutaneous booster doses of purified urease plus alum, urease-specific IgG responses elicited by the heterologous prime-boost were at least 10-fold higher than that in mice receiving purified urease or live vectors alone. A balanced Th1/Th2 response was also observed. Most importantly, partial protection was observed after prime-boost immunization upon intragastric challenge with a mouse-adapted virulent strain of H. pylori, whereas mice vaccinated with protein and live vector alone remained unprotected (89). Vindurampulle et al. (90) also investigated a heterologous mucosal prime/parenteral boost strategy, using CVD 908-htrA expressing tetanus toxin frag C. Mice primed i.n. with CVD 908-htrA expressing frag C and boosted intramuscularly (i.m.) with tetanus toxoid mounted enhanced and more rapid antitoxin responses in comparison with mice primed and boosted with parenteral tetanus toxoid alone. A permutation of the heterologous prime-boost strategy using a DNA vaccine as the priming agent, followed by subsequent boosting with live vector, was investigated by Lasaro et al. (91,92). The DNA vaccine encoded the CfaB structural subunit of the CFA/I colonization factor expressed by ETEC. The boosting agent consisted of an attenuated S. Typhimurium strain in which CfaB was expressed constitutively in the cytoplasm from a plasmid using the Ptac promoter. Mice primed i.m. with a single dose of DNA vaccine and boosted orally with the live vector developed antigen-specific mucosal IgA and serum IgG responses, which were not observed in mice immunized with DNA vaccine or live vector alone. A closer examination of the factors influencing the magnitude of responses generated revealed that the interval between priming and boosting had the strongest influence, with serum IgG titers peaking with an eight-week interval between priming and boosting and fecal IgA responses peaking with a four-week interval. Notably, anamnestic IgG responses could be elicited as long as 52 weeks after priming. CMI was shown to be tilted toward a Th1-type response, with strong IFN-g responses in the absence of IL-4. Live vectors can also successfully prime the immune system to further respond to a DNA vaccine boost. In a recent study, the plasmid-based ClyA antigen export strategy was used to improve the immunogenicity of a problematic eukaryotic antigen derived from P. falciparum (47). Mice were primed

i.n. with CVD 908-htrA exporting ClyA fused to a truncated version of the P. falciparum CSP expressed by the sporozoite stage of the P. falciparum parasite. After boosting intradermally via jet injection with a DNA vaccine encoding a eukaryotic codon-optimized version of CSP, mice developed both CSPspecific IFN-g-secreting cells and IgG capable of recognizing the native CSP protein in the membrane of P. falciparum sporozoites, as shown in Figure 2. Attempts to express unfused CSP within the cytoplasm of the S. Typhi live vector were unsuccessful (47). This report exemplifies a successful combination of refinements in expression technology, use of different vaccine formulations, and improved immunization strategies to induce potent immune responses against an otherwise poorly immunogenic antigen. In an interesting twist, use of Salmonella live vector vaccines in heterologous prime-boost strategies has been demonstrated to overcome the immature immune system of young hosts and effectively prime the neonatal immune system for potent anamnestic responses to a parenteral antigen boost given later in life (93). The stage was set when Capozzo et al. (94) demonstrated that two 109 cfu doses of CVD 908-htrA expressing frag C of tetanus administered to neonatal mice at 7 and 22 days after birth could elicit serum tetanus antitoxin responses in mice even when they were offsprings of tetanus toxoid–immunized mothers and the infants had maternal antibodies to tetanus toxin. Expanding on this observation, Ramirez et al. (93) primed newborn mice intranasally with a single dose of S. Typhi expressing Y. pestis F1 antigen and detected mucosal antibodies and IFN-g-secreting cells one week after immunization. The mice later developed a potent and rapid anamnestic response to a subsequent parenteral boost with F1-alum, which surpassed that of newborns primed and boosted only with F1-alum or S. Typhi delivering F1. Neonatal priming with F1-expressing live vector, as opposed to priming with F1-alum, resulted in a more balanced IgG2a/IgG1 profile, enhanced avidity maturation and stimulation of B memory cells, and strong Th1-type CMI. This is a promising approach to immunizing a population unable to respond to conventional vaccines early in life.

LIVE VECTOR DELIVERY OF DNA VACCINES Thus far, we have discussed the use of DNA vaccines in connection with heterologous prime-boost strategies, administered by needle and syringe or jet injection. However, DNA vaccines can also be introduced into eukaryotic target cells by invading attenuated intracellular bacteria such as Salmonella and Shigella. Although this phenomenon was first reported over a decade ago by Sizemore et al. (95,96), the exact mechanisms involved in DNA delivery and immunological priming remain unknown, and this lack of understanding has hampered efforts to improve the efficiency of the technique (97). Here, we will briefly summarize the series of events believed necessary for DNA vaccines to generate responses against a target antigen after intracellular delivery by a live vector. This initial discussion will provide a context in which recent improvements in live vector delivery of DNA vaccines can be appreciated. For a more detailed analysis of refinements in the DNA vaccines themselves and improvements to the mechanical delivery of these improved plasmids, the reader is referred to chapter 37, ‘‘DNA Vaccines.’’ For successful delivery of a DNA vaccine by an attenuated live vector, the live vector must first be endocytosed by host

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Figure 2 Immune responses in mice primed with Salmonella Typhi live vector CVD 908-htrA expressing cytolysin A–tCSP fusion protein, and boosted with the DNA vaccine plasmid pVR2571 expressing full-length PfCSP. (A) Serum IgG titers specific for the repeating NANP amino acid region of PfCSP. Arrows indicate each immunization, with the last arrow representing the boost with DNA vaccine. Solid circles represent antibody titers of individual mice, and antibody production curves are plotted on geometric mean titers. (B) Frequency of PfCSPspecific interferon-g spot-forming cells, as measured by the ELISPOT assay. Error bars indicate standard deviations. (C) Binding of PfCSPspecific serum IgG antibodies from mice, recognizing native PfCSP on the surface of Plasmodium falciparum sporozoites. Antibodies were detected by an immunofluorescence assay using FITC-labeled anti-mouse IgG, and parasite nuclei were stained with DAPI. Monoclonal antibody (MAb) 2A10, recognizing PfCSP, was used as a positive control. Source: From Ref. 47. Abbreviations: CSP, circumsporozoite protein; PfCSP, circumsporozoite protein from P. falciparum; ELISPOT, enzyme-linked immunospot assay; FITC, fluorescein isothiocyanate; DAPI, 40 , 60 -diamidino-2-phenylindole dihydrochloride.

APC. The classical APC required for initiating an adaptive immune response are dendritic cells and macrophages. After endocytosis, invading bacteria either lyse within the phagosome to release their DNA vaccines or escape from the phagosome to release their cargo directly into the cytoplasm (98). Despite the fact that Salmonella remains in the phagosomal compartment and does not escape into the cytoplasm as does Listeria or Shigella, it has now been repeatedly demonstrated that Salmonella-mediated delivery of DNA vaccines to the host cell nucleus is not only feasible but can also elicit immune responses against DNA-encoded antigens. In fact, Salmonella delivering these plasmid vaccines have been shown to induce CMI and humoral immune responses against bacterial pathogens (99) and their toxins (100), viruses (101,102), and tumor antigens (103,104). Interestingly, Salmonella-mediated delivery

of DNA vaccines can, in some cases, be more effective at inducing mucosal CMI than parenteral immunization with purified DNA (101), particularly in the context of a heterologous prime-boost strategy (47). Salmonella-DNA delivery can even be more efficient at induction of both CMI and humoral systemic immune responses when compared with prokaryotic antigen expression (100). The efficiency of plasmid transfer from live vectors to the target cell will obviously depend on the genetic stability and retention of the DNA vaccine within the live vector. Initial efforts at bacteria-mediated transfer of DNA vaccines utilized high copy number plasmids derived from pUC replicons. However, pUC-based plasmids proved notoriously unstable within live vectors, being rapidly lost from bacteria both in vitro and in vivo (99,105), and, not surprisingly, failed to elicit

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antigen-specific immunity. Interestingly, first Bauer et al. (99) and later Gahan et al. (105) demonstrated that if the copy number of the DNA vaccine within S. Typhimurium live vectors becomes too low, antigen-specific immunity is also reduced. Bauer et al. concluded that a balance is required between plasmid stability and the delivery of a sufficient amount of DNA vaccine into the phagosomal compartment to achieve bacteria-mediated genetic vaccination (99). Implicit in successful genetic vaccination using live vectors is the efficient release of cytoplasmic contents (including a resident DNA plasmid) after rupture of the cell wall. Efficient release of DNA vaccines from dying live vectors can be assured by inducing programmed bacterial lysis. Jain and Mekalanos (106) showed that the incorporation of lambda phage S and R genes into an inducible lysis system for S. Typhimurium allows for fine regulation of bacterial lysis within mucosal tissue or APC and more efficient release of DNA vaccines. Intriguingly, Loessner et al. (107) showed that the ability of an attenuated Salmonella strain to physically lyse can depend on the nature of individual mutations present in the attenuated strain. When additional mutations in either thyA (a key enzyme in DNA synthesis) or asd (essential for bacterial cell wall synthesis) were introduced into an S. Typhimurium aroA strain, removal of the required metabolite from the growth medium inactivated both attenuated strains as expected. However, only the Dasd strain lysed under nonpermissive conditions, whereas the DthyA strain remained physically intact. Release of protein and intact plasmid DNA into the surrounding medium was also conclusively demonstrated with dying Dasd strains, while no such releases occurred with dying DthyA strains. Although very promising, these improved lytic mechanisms have yet to be proven useful in vivo to enhance genetic immunization using bacterial carriers. Clearly, the release of a DNA vaccine directly into the cell cytoplasm rather than within the phagosome will be much more efficient at facilitating the steps that would eventually lead to plasmid transcription and synthesis of the encoded antigen. In this context, Gentschev et al. constructed a Salmonella strain equipped with a LLO secretion system from L. monocytogenes, which disrupts the phagosomal membrane and releases the Salmonella into the cytosol; this construct exhibited an enhanced ability to transfect mammalian cells (108). DNA delivery via recombinant Salmonella strains equipped with phagosomal escape properties was successfully used to direct macrophage presentation of a DNA-encoded antigen within MHC I and stimulate specific CD8þ T cells in vitro (109). The need for plasmid migration into the nucleus for antigen expression has been circumvented using DNA vaccine backbones derived from eukaryotic viruses that encode their own polymerases to allow direct synthesis and translation of messenger RNA within the host cell cytoplasm. This strategy was applied to the development of a DNA vaccine against measles that contained the nonstructural protein gene sequences from a human Sindbis virus that would allow direct cytoplasmic amplification of RNA encoding the measles hemagglutin and fusion proteins. When Sindbis virus–derived DNA vaccines encoding the H antigen were introduced into S. Typhi and administered to cotton rats, substantial titers of plaque reduction neutralizing measles antibodies were elicited and the animals were protected against respiratory challenge with wild measles virus (110). Considerable attention has been given to the use of Salmonella carrying DNA vaccines encoding tumor-associated antigens and cytokines as cancer vaccines. The use of bacterial

carriers that can preferentially invade tumors cells through tissue-specific colonization may allow targeted delivery of vaccine plasmids directly into cancer cells. A number of animal studies have shown that attenuated S. Typhimurium strains can successfully deliver a variety of engineered DNA vaccine plasmids for therapeutic vaccination against tumor models (111–115). CMI to tumor antigens and determinants of bacterial origin cross-presented by tumor cells appear to be the main adaptive effector mechanism contributing to tumor regression generated by these vaccines (111,116). In addition, an overall immune stimulation and a Th1-type proinflammatory environment created by the bacteria might be responsible for the success of this approach (116,117). The immune privilege of the tumor microenvironment appears to facilitate bacterial invasion contributing to the success of the live vector gene delivery and killing of infected cancer cells (116). However, the clinical effectiveness of these approaches remains to be demonstrated.

CONCLUSIONS In this chapter, we have summarized recent innovations in the use of live attenuated strains of S. enterica as live vector vaccine strains, delivering either vector-synthesized antigens or DNA vaccines to the immune system. Significant improvements in vaccination strategies have also been presented. In principle, many of the genetic techniques and strategies discussed in this chapter can contribute to the success of live vector immunization in both preclinical studies and future clinical trials.

ACKNOWLEDGMENTS This paper includes work funded by NIAID, NIH, DHHS federal research grants, contracts and cooperative research agreements: R01-AI029471, R01-AI40297, U54-AI57168, and NO1-AI25461 (to M. Levine), R01-AI36525 and NO1-AI30028 (to M. Sztein), and R01-AI065760 (to M. Pasetti). We gratefully acknowledge the expert assistance of Magaly Chinchilla.

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37 DNA Vaccines Gary J. Nabel Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

David C. Kaslow Merck, North Wales, Pennsylvania, U.S.A.

Jeffrey B. Ulmer Novartis Vaccines and Diagnostics, Cambridge, Massachusetts, U.S.A.

Margaret A. Liu ProTherImmune, Lafayette, California, U.S.A.; and Karolinska Institutet, Stockholm, Sweden

INTRODUCTION

Cellular Immune Responses

DNA vaccines have shown increasing promise as a potential vaccine modality in recent years. Having shown efficacy in preventing a variety of infectious diseases in preclinical animal models, as well as having applications to cancer immunotherapies, their potential utility in human and veterinary medicine is being realized. Three DNA vaccines have now been licensed for veterinary applications (vide infra). In humans, despite limited immunogenicity in early clinical trials, improvements in expression vectors, production technology and delivery have increased the efficacy of this technology, and its appeal has grown. Several factors have advanced this effort, including the ease of making the DNA plasmids to evaluate the protective role of different antigens for specific pathogens, the ability to more accurately and consistently measure humoral and cellular immunity in humans (including both helper and cytolytic T cells for the latter), the ease of manufacture (which has implications for vaccines needed on a global scale), and the facility of combining the DNA vaccines with other technologies such as delivery devices and adjuvants. This chapter will review the properties of DNA vaccines, giving examples of the various protective immune responses, then provide an update on advances in the technology which are leading to increased potency of DNA vaccines either used by themselves or in conjunction with other vaccine modalities or technologies. DNA vaccines are comprised of plasmids of DNA encoding antigens. These antigens can be from pathogens, tumors, or even host proteins, in the case of vaccines for the therapy of autoimmune diseases. They thus differ from traditional vaccines in not being made of an actual pathogen or of a purified or recombinant protein of a pathogen, and by being synthesized in vivo in the vaccinee following immunization. The plasmids utilize a promoter active in the recipient rather than a bacterial promoter. This in situ synthesis of the antigen results in potential advantages from a vaccine standpoint for both humoral and cellular immune responses.

Indeed, DNA vaccines were initially developed because by enabling a cell to produce an antigen in the cell, the antigen can then enter the major histocompatibility complex (MHC) class I processing pathway (which exogenously given protein antigens usually cannot) with the resultant generation of MHC class I-restricted CD8þ T cells. As these cytolytic T-cell responses are thought to play a crucial role in the defense against, and recovery from, diseases due to viruses, tumors, and certain parasites and intracellular pathogens, many vaccines under development are focusing upon technologies that can stimulate such cellular responses. Demonstration of the ability of DNA vaccines to generate effective cellular immune responses in vivo provided the first proof of the capabilities of the technology (1) by showing that a DNA vaccine encoding the internal conserved nucleoprotein of influenza virus could protect mice against death following challenge with a heterosubtypic strain of influenza. Cytolytic T-cell responses have been generated in both preclinical and human clinical studies for various pathogen and tumor antigens. Early preclinical and clinical studies with plasmids encoding proteins from a variety of pathogens and tumors including HIV (2–8), rabies (9), hepatitis B (10,11), malaria (12–15), and various tumors (11,16,17) rapidly demonstrated this capability broadly for a variety of antigens. Helper T-cell responses have also been generated by DNA vaccines, and this aspect has provided additional intriguing and potentially useful characteristics, in that DNA vaccines injected directly into muscle appear to generate Th1-biased helper responses (18,19), whereas delivery of the DNA by particlemediated bombardment (i.e., a so-called gene gun where the DNA is coated onto gold beads, and the beads are propelled into the skin) can augment Th-2 responses (20–22) As Th-1-like responses are thought to be the desired phenotype for certain vaccines, such as for HIV, Tb, and autoimmune diseases, this characteristic of DNA vaccines may be particularly important.

Chapter 37: DNA Vaccines

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Humoral Responses

Figure 1 Possible mechanisms for stimulation of adaptive immune responses by DNA vaccination. (A) Injection of plasmids encoding selective viral or microbial gene products leads to intramuscular deposition, where plasmid can be taken up by a variety of cell types, including myocytes, DC, macrophages and fibroblasts. (B) Plasmids can locally transfect DC or myocytes. Immature DCs can subsequently mature into effective antigen-presenting cells or can endocytose proteins expressed in other cells types, such as myocytes, and present them to T cells, thus stimulating CD4 and CD8 responses. Abbreviation: DC, dendritic cell.

The mechanism whereby DNA vaccines elicit the cellular responses may occur via cross-priming following transduction of nonantigen-presenting cells (23–28), as well as via direct transfection of antigen presenting cells APCs (29) (Fig. 1B). The extent of utilization of these two pathways varies depending upon the method of inoculation formulation and targeting of the DNA, which would determine which cells primarily take up the DNA and hence which produce antigen (e.g., APC vs. myocyte).

One potential difference for DNA vaccines for humoral immune responses in comparison to recombinantly made antigens is that antigens made in situ following immunization with plasmid DNA will have mammalian post-translational modifications such as glycosylation whereas antigens produced recombinantly in bacteria, yeast, or baculoviruses may not have these same modifications and hence may differ antigenically from the pathogen’s version. Of course if the antigen is one from an intracellular pathogen such as M. tuberculosis, the posttranslational modifications would not normally be mammalian, so this would not provide an advantage. But for a large number of antigens, this mammalian post-translational processing ensures that the vaccine antigen is more like the native version. Another advantage is for antigens such as the HIV Envelope (Env), which is a trimeric molecule in which each subunit is comprised of two portions, gp120 and gp41, with the latter being transmembrane (30–33). Recombinant versions initially utilized just the monomeric gp120, which not only excluded some of the epitopes most conserved between strains, found in gp41, but which could not form the trimer, which likewise has other critical epitopes (34–36). DNA vaccines can encode either full-length gp160 or truncated trimeric molecules (gp140) and elicit antibodies and T-cell responses (2). DNA vaccines were shown capable of inducing protective antibodies in preclinical models against a variety of antigens, including the initial demonstration of antibodies against hemagglutinin of influenza, which, as predicted, could protect against a homologous but not a heterosubtypic challenge (1,37–42). DNA vaccines have been shown capable of generating humoral immune responses against a large number of antigens including those derived from HIV (vide supra), rabies virus (9,43–46), and hepatitis B (11,20,22,47,48). Interestingly, the breadth of immune response as determined by the ability of the immunized animal to generate antibodies to a slightly different antigen upon secondary immunization was better for DNA encoding influenza HA than for a formalin-inactivated viral vaccine (49). Moreover, the subclass of antibodies induced by a DNA vaccine given intramuscularly (IM) encoding influenza NP or hepatitis B surface antigen reflected a more Th-1 type of T-cell help, with higher IgG2a versus IgG1 (39) and with increased production of IFN-g and lower levels of IL-4 compared to the respective licensed vaccines (50,51). However, DNA vaccine delivery via particle-mediated bombardment resulted in more IgG1 and IL-4 with less IFN-g, that is, a more balanced Th-1/Th-2 response (51,52).

TECHNOLOGIES TO INCREASE DNA VACCINE POTENCY A multipronged effort is being made to increase the potency of DNA vaccines by systematically addressing each component of the vaccines and each stage in the delivery and immunogenicity-inducing process. The plasmids have been constructed to express more protein using techniques such as codon optimization, employing codons for which more mammalian t-RNA exists compared to the codons which a pathogen may have utilized (53,54). Additional approaches have compared leader sequences, used different transcriptional control elements (55) and employed higher-expressing promoters (56). Updates on other approaches will be described in more detail below.

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DNA Vaccine Formulation and Delivery By the use of fluorescently tagged plasmid DNA, Dupuis and colleagues (29) showed that most of the DNA did not transduce cells and result in protein expression, confirming the low levels of gene expression shown by Felgner and colleagues in their initial publication (57) showing that muscle cells could take up plasmid and express the encoded protein. Efforts to increase the amount of DNA delivered into cells have focused on formulating the DNA in order to increase the amount taken up by cells (and decrease degradation of the DNA) or utilizing devices to increase cellular transduction. Formulations The initial demonstration of the ability of plasmid DNA to be taken up by muscle cells and to express their encoded protein following IM injection, was actually a control for an experiment using plasmid formulated in cationic lipids. It was surprising that the ‘‘control’’ of the unformulated DNA resulted in more effective uptake and expression than the formulated DNA. Nevertheless, more recent formulations have proven to have increased efficacy compared to unformulated, ‘‘naked’’ DNA. These formulations are designed to protect the DNA from nuclease digestion, to increase the uptake of DNA into cells, to deliver the DNA mucosally, to help target the DNA to specific receptors or cells, or to provide a means to attach adjuvants. One such vaccine formulation appears to itself provide adjuvant activity for the immune responses against influenza (58,59). DNA has been formulated into microparticles to take advantage of the ability of APCs to take up particles sized approximately 1 to 10 mm in diameter (60–62). The DNA has been either placed inside the particle (62–66) or adsorbed on the surface (67–70). Cationic liposomes (71), block copolymers (72,73), polyethyleneimine (74), polylysine (75), and virus-like particles (76,77) are all examples of these promising formulations for DNA vaccines, with poly(D,L-lactic-co-glycolic acid) (PLG) being used for both systemic and mucosal administration (78). Devices that propel the DNA either in liquid form or formulated onto gold beads have been used in both preclinical and clinical trials (20–22,79). The gene-gun technology (where the DNA is coated onto gold beads) has proven to be more efficient for the generation of immune responses compared to IM injection, even generating antibody responses against hepatitis B surface antigen in patients who are nonresponders to conventional vaccine (48), but is limited by the dose that can be delivered, thus requiring multiple injections and sites. Other devices, such as the Biojector, directly propel under high pressure a stream of liquid intradermally (79–81). Electroporation, in which an electric field is applied to the tissue after the injection of the DNA, greatly increases the uptake of DNA into cells (82–85). Many more cells thus produce antigen, and the resultant immune responses are significantly greater in preclinical studies in mice, rabbits, and nonhuman primates. Because electroporation increases the number of cells transduced as well as the amount of plasmid in nuclei, concerns exist that the risk of integration may increase (86). This issue is being carefully evaluated. Another issue for vaccines for diseases such as HIV is global affordability and access for a vaccine that requires the use of a specialized delivery device.

Mucosal and Transcutaneous Administration The transcutaneous route of immunization is being evaluated for DNA vaccines as for other vaccine formulations (87,88). This

approach has shown efficacy in a preclinical SIV/HIV model (89) and an influenza model (90). Mucosal administration (intranasal or oral) is appealing both because the route may be more amenable for usage in resource-poor settings, and because of the hopes of raising mucosal immune responses. DNA vaccines have been delivered mucosally either in formulations or utilizing devices (60,91–93).

Prime-Boost (Mixed Modality) Vaccination In a challenge model of malaria, DNA immunization followed by boosting with a viral vector that encoded the same malarial antigen resulted in both greater immune responses and improved protection compared to either vector alone, or the reverse order of the viral vector given before the DNA (94). This observation, referred to as ‘‘prime-boost’’ or ‘‘mixed-modality’’ immunization, has been repeated in other preclinical disease models and in early human clinical trials (81,95—100) and in studies when the DNA vaccine is followed by immunization with other viral vectors or recombinant protein (85,101) The mechanism for this increased potency is not fully understood, but because it appears to consistently work best when the DNA vaccine is given first, it is thought that not having other viral antigens present during the initial immunization may play a role. The rationale for utilizing a recombinant protein as a boost was to increase the antibody response, which has not been very potent in primate models or humans when using a DNA vaccine. Interestingly, in a prime-boost study with an HIV protein, the recombinant protein boost resulted in stronger cellular as well as humoral responses (102). Prime-boost strategies have been combined with formulated DNA as well. As an example, increased helper T-cell production of IFN-g and production of neutralizing antibodies were seen following a protein boost after priming with PLGmicroparticle-formulated DNA (85).

Adjuvants and Cytokines Coadministration of cytokines (either as recombinant proteins or DNA plasmids) has been effective for increasing the potency of DNA vaccines, either for DNA vaccines as a single modality or in prime-boost regimens (reviewed in Refs. 103–109). A variant on the approach of co-injecting plasmids encoding the cytokine and the antigen was to make a single plasmid encoding a fusion protein of antigen (HIV Env gp120) linked to IFN-g (110). This highlights the flexibility of the technology. IL-15 given as a codon-optimized plasmid increased CD8þ T cells when given with an HIV gag DNA construct (111). Other constructs encoding IL-10 (112), IL-18 (113), and the chemokine MIP-1 a (114) have been evaluated. Recombinant cytokines have also been utilized, but the rationale for giving the cytokine as a gene rather than recombinantly relates to the kinetics and location of the antigen production.

Innate Immunity DNA plasmids are produced in bacteria, and their backbones, in contrast to mammalian DNA, contain unmethylated CpG motifs, which stimulate the innate immune system by signaling through Toll-like receptor (TLR) 9 (115–118). Thus, some of the immunogenicity of DNA vaccines may be due to this intrinsic structure function relationship. Addition of plasmid that did not encode the relevant antigen was shown to augment the antigen-specific immune responses (49,115,119,120). However, DNA vaccine immunization of TLR9-/-mice demonstrated little or no role

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for TLR9 signaling in the adaptive immune response against the encoded antigen (121–123). Nevertheless, in part because synthetic CpG oligonucleotides appear to function as adjuvants for proteins (124), efforts have been made to alter the backbone of the plasmids to encode more CpG motifs in an effort to increase the immunogenicity of DNA vaccines.

Novel DNA Constructs Beyond double-stranded bacterial plasmid DNA that requires the use of antibiotic resistance genes and other sequences unrelated to those for eliciting protective immunity, future applications may include linear DNA-based vaccines that have been rapidly manufactured in cell-free systems. One form of these enzymatically produced linear expression cassettes, when formulated with cationic lipids and administered once at low microgram amounts of DNA, has recently been shown to elicit high level protective immunity against homotypic influenza challenge in mice (125). Manufacture of these vaccines is more akin to a synthetic small molecule process, and as such, may provide an alternative rapid, scalable process advantageous in an emergent or pandemic situation.

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immunity in fish may be the result of early innate immune stimulation triggered by nonspecific G protein effects, followed by long-term memory immunity to G (132). The mode of action of the IHNV DNA vaccine in fish has not been fully elucidated but may involve both neutralizing antibodies and T-cell responses (133), as salmon have a relatively full repertoire of Th1 cytokines and functional cytotoxic T lymphocytes (134).

West Nile Virus for Horses A DNA vaccine encoding the PreM-E antigen of WNV was licensed for use in horses in July 2005. Preregistration data demonstrated strong potency and efficacy in several animal models. In mice, WNV DNA induced neutralizing antibodies and protective efficacy against lethal live virus challenge (135). In birds, a single intramuscular dose of WNV DNA conferred protection against live WNV challenge, as measured by survival and lower viremia (136). In horses, WNV DNA was immunogenic, as judged by the induction of neutralizing antibodies, and conferred protection against live virus challenge, as measured by reductions in viremia and progression to clinical disease (135). These encouraging data have lead to the evaluation of a similar vaccine in human clinical trials (vide infra).

Melanoma for Dogs

The recent licensure of three DNA vaccines for animal health applications has prompted renewed interest in the broader potential of this technology. Within two days of each other in July 2005, the first DNA vaccines to obtain licensure were ones to prevent disease caused by infectious hematopoietic necrosis virus (IHNV) in farm raised Atlantic salmon (Apex-IHN1, Novartis Animal Health, Basel, Switzerland) and West Nile virus (WNV) in horses (Fort Dodge Laboratories, Fort Dodge, Iowa). In March 2007, a therapeutic DNA vaccine to treat melanoma in dogs (Merial) received conditional approval from the U.S. Department of Agriculture. Thus, the total successes to date include two preventative vaccines for infectious diseases and a therapeutic vaccine for cancer. At the very least, this will spur efforts to understand the reasons behind these successes and whether this information can be used to enable DNA vaccines for humans.

A therapeutic DNA vaccine encoding human tyrosinase to treat malignant melanoma in dogs received conditional licensure in March 2007. This xenogeneic approach to breaking tolerance has proven to be very effective in preregistration studies in dogs (137–139). In a phase 1 trial, dogs with stage II, III or IV malignant melanoma were treated with tyrosinase DNA biweekly for four weeks. The vaccine appeared efficacious, based on clinical antitumor responses and survival time, versus historical controls treated with surgery only (137). In expanded studies, long-term survival (>1600 days) has been observed, even in DNA vaccine-treated dogs with advanced stages of disease (138,139). A preliminary investigation of mechanism of action suggests that onset of anti-tyrosinase antibodies correlates with clinical responses (139). These compelling data in dogs support evaluation of tyrosinase DNA vaccines for treatment of melanoma in humans.

Infectious Hematopoietic Necrosis Virus for Fish

HUMAN VACCINE TRIALS

Apex-IHN to target IHN in fish was approved for market by the Canadian Food Inspection Agency, Veterinary Biologics and Biotechnology Division (VBBS), in July 2005. IHNV is a single-stranded negative sense RNA virus (family Rhabdoviridae) that infects wild and farmed salmonid fish in the Pacific Northwest of North America and causes an extensive necrosis of hematopoietic tissues in early life stages. Neutralizing antibodies directed toward the viral surface glycoprotein (G) (126) confer protection against disease, while other viral proteins did not appear to be targets of protective immunity (127). Field trials have demonstrated that a single inoculation of a low dose (10 mg) of IHNV DNA vaccine encoding the G protein provides long-lived immunity to the disease (128). The specific reason(s) for the effectiveness of this vaccine remains to be determined, but does not appear to be a general phenomenon of DNA vaccines in fish (129). Rather, the inherent antigenicity of the antigen itself may be important. For example, the G protein of rhabdoviruses, including IHNV, can induce rapid type I interferon production by cells (130,131). Therefore, antiviral

The efficacy of DNA vaccines has been demonstrated in a variety of animal models. The utility of this approach has recently undergone further evaluation in human clinical trials (Table 1). Although initial studies suggested that the immunogenicity of DNA vaccines in humans may not be sufficient to generate immune responses, several studies now suggest that immunogenicity can be achieved in a variety of infectious disease models. Initial evidence for the in vivo effects of DNA vaccination was described in malaria DNA vaccines (12,14). T-cell responses were demonstrated, though they were relatively low in magnitude. Subsequent human studies have provided independent evidence that DNA vaccines induce immune responses in humans. These responses include not only T-cell immunity but also the generation of antibodies and, in some instances, neutralizing antibody responses. A study using plasmid DNAs encoding multiple gene products from HIV, including Gag, Pol, Nef, and Envs from clades A, B, and C showed the ability to elicit T-cell responses, as measured by both ELIspot analyses

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Table 1 Summary of Clinical Studies of DNA Vaccines for Various Disease Targets with Indicated Genes Target

Agent

Gene insert

Viral infectious disease

Hepatitis

Hepatitis B surface antigen (HBsAg) Hepatitis B mixed plasmid DNA-110 (HB-110) Herpes simplex virus type 2 (HSV-2) gene fragment Regulator of virion protein expression (Rev), transactivator of transcription (Tat), or negative regulatory factor (Nef) genes HIV-1 envelope gene (Env) and Rev Clade A env (VRC-HIVDNA044-00-VP) HIV-1 associated encephalopathy (-AE) antigens, modified group antigen gene (Gag), polymerase gene (Pol), Tat/Rev and Env Polyepitopic: Gag, Pol, viral protein R (Vpr), Nef, Rev, Env (EP HIV-1233) Clade B Gag, Pol, Nef; Clade A,B,C Env (VRC-HIVDNA016-00-VP) (VRC-HIVDNA-009) Clade B Gag, Pro, RT, Env, Tat, Rev, viral protein U (Vpu) HIV-1 Gag Polyepitopic: Gag, Pol, Vpr, Nef (EP HIV-1090) Clade B Env, Gag, (reverse transcriptase) RT, Tat, Vpu, Rev Env, Rev (APL 400-003) Clade B Gag (gag-2962) Clade B Gag, Env HIV DNA gag/multiepitope (pTHr.HIVA) Clade A-derived p24/p17 gag fused to a string of HLA class I epitopes Clade B Gag, Pol, Env (PENNVAX-B) Clade C Gag, RT, tat, Nef; clade C Env (SAAVI DNA-C2) Influenza virus H5 hemagglutinin protein (VRC-AVIDNA036-00-VP) Trivalent DNA vaccine (PIA0601) Histone H5 (pPML7789) Spike (S) protein of SARS coronavirus (CoV) (VRC-SRSDNA015-00-VP)

Herpes simplex virus Human immunodeficiency virus (HIV)

Influenza

Severe acute respiratory syndrome (SARS) West Nile virus (WNV)

Parasitic infectious disease Cancer

Allergic disease

Measles virus Malaria Malaria B-cell lymphoma Breast cancer ovarian cancer Cervical cancer precancerous/ nonmalignant condition Liver cancer Melanoma Melanoma Melanoma Melanoma (skin) intraocular melanoma Melanoma (skin) intraocular melanoma Prostate cancer Prostate cancer Prostate cancer bladder cancer non–small cell lung cancer esophageal cancer sarcoma Ragweed allergen

WNV viral protein precursor transmembrane (prM) and Env (VRCWNVDNA020/17-00-VP) Measles hemagglutinin (H) alone or H plus fusion (F) protein Plasmodium falciparum multiple epitope (ME)-thrombospondin-related adhesion protein (TRAP) (ME-TRAP) Plasmodium falciparum circumsporozoite protein (PfCSP) Tumor heavy- and light-chain variable region HER-2/neu intracellular domain (pNGVL3-hICD) Detox form of the human papillomavirus type 16 (HPV-16) antigen E7 a-Fetoprotein Melanocyte differentiation antigen (MART-1) and HBsAg Glycoprotein-75 (gp75) Tyrosinase 207–216 and tyrosinase 1–17 (synchotrope TA2MTM) Xenogeneic tyrosinase Mouse glycoprotein-100 (gp100) Prostate-specific membrane antigen (PSMA), CD86, PSMA/CD86 Prostate-specific antigen (PSA) Cancer-testis antigen (NY-ESO-1)

Ragweed-pollen antigen (Amb a 1)

and intracellular cytokine responses (140). The T-cell responses by ELIspot were approximately 80%, with CD4 responses by flow cytometric intracellular cytokine staining (ICS) approaching 100%, particularly for the clade A Env immunogen. CD8 responses were in the 40% –to 50% range. The T-cell responses were persistent, measurable for more than one year after the initial immunization, and a significant percentage of subjects were found to synthesize antibodies reactive with the Env immunogens in the vaccine.

Subsequent vaccine studies have shown that these DNAs are able to prime responses for a recombinant rAd boost, and the immunogenicity of the prime-boost combination exceeded that of each component alone (unpublished observations). On the basis of these studies, phase II trials of a DNA prime-Ad boost vaccine for HIV have now been completed, and further, an efficacy study is planned (Fig. 2). In addition to the HIV vaccine studies, DNA vaccines have been tested for their ability to elicit immune responses

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suggest that it is possible to generated SARS virus immunity with plasmid DNA encoding the S protein of this new virus, previously shown in mice to be protective against viral challenge (144). Such a vaccine was able to elicit neutralizing antibodies in this model. The difficulty in advancing a SARS vaccine to licensure lies in the lack of a relevant animal model that mimics human disease and the absence of active disease in which vaccine efficacy can be tested.

CONCLUSIONS AND PROSPECTS Figure 2 Design of the first human efficacy study of an AIDS vaccine which includes a plasmid DNA component. The immunization schedule and major features of the study design are indicated.

against other pathogens. Following the success of a DNA prime-Ad boost for Ebola virus in nonhuman primates (141), the components for this vaccine have now advanced into human testing. A phase I study of Ebola DNAs encoding the glycoprotein of the Zaire and Sudan strains, as well as the nucleoprotein, has been conducted (142). Similar to the HIV studies, the majority of patients generated ELIspot and CD4 responses (90–100%) with a response rate of approximately 30% for CD8 responses by ICS. In addition, antibodies specific for each component of the vaccine were detectable by immunoprecipitation followed by Western blotting. In this instance, the frequency of these positive responses seemed highest at the highest dose of vaccine, 8 mg, although 4 mg responses were similar. The 4 mg dose is planned for future development, for which replication-defective rAd boosting with GPs is planned. A vaccine to protect transplant patients from CMV-associated disease is yet another plasmid DNA-based effort, which has now reached Phase II clinical studies. The bivalent vaccine, VCL-CB01, contains two plasmids, one encoding CMV phosphoprotein 65 (pp65) and another glycoprotein B (gB), for induction of cellular and humoral immune responses, respectively. The plasmids are formulated with poloxamer CRL1005 and benzalkonium chloride to enhance immune responses. In healthy CMV seronegative adult subjects, 1 mg or 5 mg doses of vaccine administered intramuscularly on a 0-, 2-, and 8-week schedule, or 5 mg doses of vaccine administered on a 0-, 3-, 7-, and 28-day schedule, were well tolerated and elicited gB antibodies and T-cell responses to both pp65 and gB at 1 mg or 5 mg on either injection schedule (143). More recently, it has been possible to generate neutralizing antibodies after DNA vaccination in humans. This response was generated after vaccination with a DNA vaccine encoding West Nile Virus, particularly the core, pre-M and E vectors. This vaccine was based on a WNV vaccine that protected in murine challenges (135), which subsequently has received regulatory approval for use in animals (vide supra). In the human study, neutralizing antibody responses were generated in addition to cell-mediated immunity, as measured by CD4 and CD8 ICS. Interestingly, these antibodies neutralized effectively and at levels that were comparable to horses, for which the animal vaccine was approved, with comparable titers. These data highlight the potential utility of such a vaccine, not only for humans but for animals as well. Data

A variety of technological developments have successfully increased the immunogenicity of DNA vaccines. These have included improvements in the vaccines themselves (such as increasing the expression of the encoded antigen), delivery devices, formulations, and combinations with other vaccine modalities. The licensure for veterinary use of two DNA vaccines and one DNA plasmid cancer immunotherapy provide encouragement for the ongoing preclinical efforts and clinical trials of human DNA vaccines and immunotherapies. While increased potency may still be needed, perhaps the greater issue will be whether the increased complexity of the different embodiments of the DNA vaccines will be an issue for vaccines that are needed for global diseases such as HIV. The broad application of the technology both as a research tool and for the prevention and therapy of such a broad array of diseases, and the flexibility of the technology to incorporate and adapt to novel delivery systems, adjuvants, and other immunostimulatory/immunoregulatory interventions, continue to attract interest for the technology.

ACKNOWLEDGMENTS We thank Mike Cichanowski and Brenda Hartman for graphics, Ati Tislerics for manuscript preparation, and Drs. Barney Graham and Daniel Bailey for assistance in compiling Table 1.

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107. Calarota SA, Weiner DB. Enhancement of human immunodeficiency virus type 1-DNA vaccine potency through incorporation of T-helper 1 molecular adjuvants. Immunol Rev 2004; 199:84–99. 108. Stevenson FK. DNA vaccines and adjuvants. Immunol Rev 2004; 199:5–8. 109. Barouch DH. Rational design of gene-based vaccines. J Pathol 2006; 208:283–289. 110. Nimal S, McCormick AL, Thomas MS, et al. An interferon gamma-gp120 fusion delivered as a DNA vaccine induces enhanced priming. Vaccine 2005; 23:3984–3990. 111. Kutzler MA, Robinson TM, Chattergoon MA, et al. Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help. J Immunol 2005; 175:112–123. 112. Daly LM, Johnson PA, Donnelly G, et al. Innate IL-10 promotes the induction of Th2 responses with plasmid DNA expressing HIV gp120. Vaccine 2005; 23:963–974. 113. Luo Y, Zhou H, Mizutani M, et al. A DNA vaccine targeting Fosrelated antigen 1 enhanced by IL-18 induces long-lived T-cell memory against tumor recurrence. Cancer Res 2005; 65:3419–3427. 114. Sumida SM, McKay PF, Truitt DM, et al. Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines. J Clin Invest 2004; 114:1334–1342. 115. Hemmi H, Takeuchi O, Kawai T, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408:740–745. 116. Krug A, Towarowski A, Britsch S, et al. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur J Immunol 2001; 31:3026–3037. 117. Takeshita F, Leifer CA, Gursel I, et al. Cutting edge: role of Tolllike receptor 9 in CpG DNA-induced activation of human cells. J Immunol 2001; 167:3555–3558. 118. Chuang TH, Lee J, Kline L, et al. Toll-like receptor 9 mediates CpG-DNA signaling. J Leukoc Biol 2002; 71:538–544. 119. Sato Y, Roman M, Tighe H, et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 1996; 273:352–354. 120. Klinman DM, Yamshchikov G, Ishigatsubo Y. Contribution of CpG motifs to the immunogenicity of DNA vaccines. J Immunol 1997; 158:3635–3639. 121. Spies B, Hochrein H, Vabulas M, et al. Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 2003; 171:5908–5912. 122. Babiuk S, Mookherjee N, Pontarollo R, et al. TLR9-/- and TLR9þ/þ mice display similar immune responses to a DNA vaccine. Immunology 2004; 113:114–120. 123. Tudor D, Dubuquoy C, Gaboriau V, et al. TLR9 pathway is involved in adjuvant effects of plasmid DNA-based vaccines. Vaccine 2005; 23:1258–1264. 124. Daubenberger CA. TLR9 agonists as adjuvants for prophylactic and therapeutic vaccines. Curr Opin Mol Ther 2007; 9:45–52. 125. Vilalta A, Jimenez G, Rusalov D, et al. Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza a challenge. Hum Gene Ther 2007; 18: 763–771. 126. Lorenzen N, Olesen NJ, Jorgensen PE. Neutralization of Egtved virus pathogenicity to cell cultures and fish by monoclonal antibodies to the viral G protein. J Gen Virol 1990; 71(pt 3): 561–567. 127. Corbeil S, Lapatra SE, Anderson ED, et al. Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout oncorhynchus mykiss using DNA vaccines. Dis Aquat Organ 1999; 39:29–36. 128. Anderson ED, Mourich DV, Fahrenkrug SC, et al. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against

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38 Overview of Heterologous Prime-Boost Immunization Strategies Helen McShane and Adrian V. S. Hill The Jenner Institute, University of Oxford, Oxford, U.K.

Andrew McMichael Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, U.K.

INTRODUCTION Three of the most important infectious pathogens throughout the world—HIV, Mycobacterium tuberculosis, and Plasmodium falciparum—are all intracellular pathogens, and protective immunity is mediated, at least in part, by the cellular arm of the immune system (1–3). Furthermore, a potent cellular immune response is critical for the control of many persistent viral infections such as HIV and hepatitis C virus (4,5). The development of vaccines against these pathogens requires vaccination strategies effective at inducing high level and persistent cellular immune responses. There are a variety of antigen delivery systems that induce a cellular immune response, including DNA vaccines, recombinant viral vectors, virus-like particles, recombinant bacteria, and protein/adjuvant combinations. However, when these vaccine candidates are used alone, it has proved difficult to induce high levels of cellular immune responses. Homologous boosting with the same vaccine often does not substantially amplify the induced cellular immune response. Heterologous prime-boost immunization regimens are where two different vaccines, each encoding the same epitope or antigen, are given at discrete intervals, ranging from weeks to months. Such regimens are highly effective at inducing higher levels of both major histocompatibility complex (MHC) class I restricted CD8þ T cells and class II restricted CD4þ T cells than homologous boosting and enhanced protection against infectious challenge in preclinical models (6–9). In addition to enhancing cellular immune responses, heterologous prime-boost regimens appear to be effective at inducing higher humoral responses as well (10,11). This may be important for those pathogens where antibodies are felt to contribute to protective immunity. In the last five years, heterologous prime-boost immunization strategies using non-replicating viral vectors as boosting agents have increasingly been evaluated in early-stage clinical trials, primarily in the field of infectious diseases but also for therapeutic vaccination against tumors. In this chapter, we will begin by discussing potential mechanisms for the enhanced immunogenicity and protective efficacy seen with heterologous prime-boost immunization strategies. We will then review the data from clinical trials to evaluate whether the promise suggested by the preclinical data

translates into what is seen in the clinical scenario, and discuss how the results of some of these clinical trials are now being taken forward into larger-scale efficacy trials.

ANTIGEN DELIVERY SYSTEMS AND ROUTE The first report demonstrating the effectiveness of heterologous prime-boost immunization strategies used a recombinant influenza virus expressing a malarial antigen as a priming agent and a recombinant vaccinia virus expressing the same antigen as the boost (6). Subsequently it was found that, surprisingly, nonreplicating vectors could induce comparable or stronger cellular immune responses than these replicating viruses (7). Since this early work, many different antigen delivery systems have been evaluated in heterologous prime-boost regimens. Plasmid DNA, recombinant protein combined with adjuvant, and recombinant viral and bacterial vectors are among those most commonly used. However, while all of these vectors can prime an immune response, some are more effective than others at boosting. In particular, recombinant viral vectors, including both poxviral vectors such as modified virus Ankara (MVA) and adenoviral vectors have been found to be extremely effective at boosting previously primed cellular immune responses (12–14). Further discussion on the individual viral vectors can be found elsewhere in this volume. The order in which the vaccines are administered may be important in the effectiveness of boosting; not all vectors are effective boosting agents. Priming with DNA and boosting with a viral vector was the most effective vaccination order for both T-cell induction and protection, in a murine model of malaria, when the vaccines were administered systemically (Fig. 1). Intriguingly, one report suggests that this is not the case when the vaccines are administered by the mucosal route, where the most effective regimen was priming with a recombinant vaccinia virus expressing the glycoprotein (gB) of herpes simplex virus and boosting with DNA (15). This finding may be explained in part by the viral vector being more potent than the DNA vaccine, but further work is needed on the importance of route in vaccination order, particularly as many of the intracellular pathogens in question have a mucosal portal of entry. Further evidence of the importance of route comes from some

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Figure 1 Immunogenicity of various prime-boost immunization regimes. The CD8 T-cell response to a nonamner Kd-restricted epitope in the circumsporozoite protein of Plasmodium berghei was measured by g-interferon enzyme-linked immunospot assay of Balb/c mouse splenocytes after various immunization regimes, shown on the x-axis legend. Source: From Ref. 7.

work on tuberculosis (TB). Priming with Bacille CalmetteGue´rin (BCG) and boosting with a recombinant adenovirus (rAd)-expressing antigen 85A is effective at enhancing protective efficacy, but only if the adenovirus (Ad) is administered by the intranasal route, not if it is administered by either intramuscular or subcutaneous routes (16).

POTENTIAL MECHANISMS There are several potential mechanisms by which heterologous prime-boost strategies enhance the cellular immune response induced. The use of different sequential vectors to deliver the antigen in question will avoid the induction of both humoral and cellular immune responses to the vector; this anti-vector immunity inhibits effective homologous boosting with the same vector. The priming of an immune response to a few immunodominant epitopes that are then preferentially boosted when a second delivery system encoding the same antigen is administered may also be important. Interestingly, there is some evidence that prime-boost regimes enhance the breadth as well as the magnitude of induced immune response. Vaccination with DNA primeadenoviral boost in mice resulted in an increased diversification of the CD4þ T-cell response, when compared with either DNA or adenoviral vaccination alone (17). In contrast, the CD8þ T-cell response was increased in magnitude but not breadth, after the prime-boost regimen. These two antigen delivery systems utilize divergent cell targeting mechanisms, and different modes of antigen processing and presentation, which may explain this differential effect. There are also vector-specific issues that explain why some vectors are particularly good at boosting. Poxviruses induce strong innate, nonspecific immune responses, which facilitate the amplification of the adaptive immune response induced (18). Interestingly, the loss of immune evasion genes in MVA may contribute to the superior boosting effect seen with MVA when compared with wild-type vaccinia (7,18). Expressionprofiling studies have revealed differences in the range of tran-

scripts induced by MVA, NYVAC (attenuated vaccinia virus) and replication-competent vaccinia strains (19,20). Finally, the induction of high avidity, as well as high frequency CD8þ T cells, may help explain the effectiveness of this approach (21). Recent data from a clinical trial of a new TB vaccine illustrates the importance of evaluating the regulatory T-cell response as well as the effector response. Boosting BCG vaccinated subjects with MVA85A resulted in a downregulation in transforming growth factor beta-1 (TGF-b1) and an increase in the enzyme-linked immunospot (ELISPOT) response to the recall antigen streptokinase/streptodornase (SK/SD) (22). In this study, there was a correlation between CD4þCD25hiFoxP3þ cells and TGF-b1 serum levels. It is possible that this downregulation of TGF-b1 accounts, at least in part, for the sustained high levels of antigen-specific effector T cells seen after vaccination with MVA85A in BCG primed subjects, perhaps through a reduction in the number of circulating regulatory T cells. The optimal interval between priming and boosting vaccination needs to be sufficient for the induction of a memory T-cell population, which can subsequently be boosted. In preclinical studies, boosting less than nine days after priming appears to be suboptimal, at least for the boosting of CD8þ T cells (12). In the clinical studies reviewed later in this chapter, the minimum interval between priming and boosting was three weeks.

ANTI-VECTOR IMMUNITY The avoidance of anti-vector immunity is one mechanism by which heterologous prime-boost regimens are more immunogenic than homologous boosting regimens. Furthermore, preexisting immunity to the vector will potentially abrogate any boosting effect. For example, preexisting immunity to human adenoviral strains varies considerably, but can be up to 80% for common human adenoviral strains such as Human serotype 5 (23). Results using a recombinant (Human serotype 5) adenoviral

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vector expressing an HIV-1 immunogen suggest that preexisting antibodies to this strain of Ad might reduce the magnitude of cellular immune response induced by more than threefold (24,25). However, in both of these clinical trials, vaccine-induced immune responses were detected, even in subjects who were seropositive for Ad5 before vaccination, and the use of higher doses of the Ad5 vaccine helped to overcome this preexisting anti-vector immunity. The utilization of less-prevalent human strains, or simian strains to which there is little human exposure, may circumvent this problem (26). The use of heterologous adenoviral subtypes from different serotypes may also help circumvent the problem of anti-vector immunity (27). Preexisting immunity against smallpox does not appear to be a problem for the use of MVA as a vector, perhaps in part because of the long time interval between smallpox vaccination and the current studies with MVA. The same viral vectors are currently being used for the development of vaccines against multiple pathogens; this may limit the number of times the same vector is utilized. There is evidence from clinical trials that anti-vector immunity against MVA is present at three weeks but lasts less than one year (28,29). Further work is needed to define more precisely the nature and length of anti-vector immunity for these promising boosting vectors.

CLINICAL TRIALS Following the success of prime-boost immunization strategies in preclinical models, many regimens have been evaluated in small-scale clinical trials. The main outcome measures used in these clinical trials are safety and immunogenicity. These data are essential in proceeding with the clinical evaluation of these strategies; however, the precise immunological correlates of protection are not clearly defined for these pathogens. For this reason, even small-scale clinical studies that evaluate efficacy, such as those conducted with vaccines against malaria and cancer, are invaluable in evaluating the potential utility of prime-boost strategies in humans, and in identifying potential correlates of protection.

Malaria Since 1999, there have been a series of clinical trials evaluating the use of several vectors expressing pre-erythrocytic antigens from P. falciparum, both alone and in combination in heterologous prime-boost regimens. These trials initially evaluated the safety and comparative immunogenicity of different heterologous prime-boost regimens. The protective efficacy of the most immunogenic regimens was subsequently evaluated using sporozoite challenge (30). The early trials used plasmid DNA as a priming agent and boosted with a recombinant MVA expressing the same antigen. The antigen used in these studies was the pre-erythrocytic protein antigen, thrombospondin-related adhesion protein (TRAP), which was fused to a polyepitope string of 14 CD8þ T-cell epitopes (multiple epitopes, ME) from six different pre-erythrocytic P. falciparum antigens (ME-TRAP). A range of DNA doses from 0.5 to 2.0 mg were evaluated in these clinical trials, and DNA ME-TRAP was also delivered using a needleless ballistic ‘‘gene gun’’ device. Recombinant MVA ME-TRAP was delivered intradermally at doses of 3  107 to 1.5  108 pfu. In total, 150 vaccinees received these vaccines and the safety profile was good for both vaccines (31). The results of these early trials demonstrated that 5- to 10-fold higher immunogenicity was seen in the heterologous

prime-boost regimes than in the homologous boosting regimes (30). These were predominantly CD4þ T cells, with fewer CD8þ T cells and little or no antibody induced. Two or three priming immunizations followed by an MVA boost intradermally at a short interval were particularly immunogenic. Lengthening the interval between the final DNA and the MVA immunization from three to eight weeks appeared to reduce immunogenicity slightly. Importantly, these immunogenic regimes were found to confer partial protection after sporozoite challenge manifest as a delay in time to parasitemia. The protective efficacy of this DNA prime-MVA boost was subsequently evaluated in a phase IIb efficacy trial in Gambian adults (32). This trial demonstrated that although vaccination with two doses of DNA ME-TRAP followed by a single boost with MVA ME-TRAP was safe and highly immunogenic for effector T-cell induction, it did not significantly reduce the P. falciparum infection rate in a semiimmune adult African population. This is unlikely to have been caused by TRAP strain variation in field parasites as T-cell responses were broad and largely cross-reactive between strains. Preclinical studies aimed at improving both immunogenicity and protective efficacy demonstrated better immunogenicity and efficacy in mice when a second viral vector, recombinant fowlpox (strain FP9), was used to prime and MVA used to boost (33). Using the same insert as in the previous studies, a clinical trial with FP9 ME-TRAP primeMVA ME-TRAP was subsequently conducted in U.K. adults. This vaccination regimen was found to be safe and well tolerated (34). Although the maximal ELISPOT level induced was lower than with the optimized DNA-MVA regime, a higher proportion of CD8þ T cells were induced with the FP9-MVA regimes (35). The protective efficacy of this regime was subsequently evaluated by sporozoite challenge, and in a small number of individuals complete sterile protection was induced that lasted for up to 20 months (36). Interestingly, protection at 20 months was associated with persisting memory but not effector T-cell responses. Importantly, for both DNAMVA and FP9-MVA the protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses (30,36,37) (Fig. 2), supporting the strategy of maximizing durable T-cell immunogenicity to develop more effective liver-stage vaccines against P. falciparum malaria. Using the percentage reduction in liver-stage parasites as a measurement of efficacy, the FP9-MVA regime produced a 92% reduction in parasite burden compared with an 80% reduction with DNA-MVA regimes (38). This FP-MVA prime-boost regimen was then evaluated in a phase I trial in Kenyan children and immunogenicity was lower than in U.K. adult vaccinees (39). This clinical study also investigated the effects of antivector immunity and found that partial cross-reactive immunity induced by administration of the first poxviral vector reduced the immunogenicity of the second, boosting vector (39). This study intriguingly suggests that priming with a lower initial dose limits this anti-vector immunity and paradoxically stimulates stronger cellular immunity after boosting than full dose priming. Further work on anti-vector immunity showed that alternating vector regimes (e.g., MVA/FP9/MVA or FP9/ MVA/FP9) induced higher levels of memory T cells, as measured in a cultured ELISPOT assay than heterologous primeboost regimens such as FP9/MVA or FP9/FP9/MVA (40). A phase IIb field trial was conducted in Kenyan children to evaluate the protective efficacy of FP9 ME-TRAP prime-MVA ME-TRAP boost regime. The immunogenicity results seen in this trial were disappointingly low, probably explaining the

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insert in a phase I trial at Oxford and stronger immune responses detected than with DNA-MVA and FP9-MVA regimes. Such Ad-MVA regimes were found to induce strong antibody responses in preclinical malaria models, suggesting that they could be of value for blood-stage malaria vaccines (11). DNA-Ad prime-boost regimes are currently planned for phase I malaria vaccine trials on the basis of promising results in nonhuman primates.

Tuberculosis

Figure 2 Correlation between ex vivo IFN-g ELISPOT responses to the METRAP malaria vaccines and days to parasitemia for a group of subjects vaccinated with DNA prime-MVA boost. The summed ex vivo IFN-g ELISPOT response one week after the MVA boost correlates with the number of days to parasitemia as measured by Spearman’s rank correlation coefficient (r ¼ 0.793, two tailed, p ¼ 0.033). Abbreviations: ELISPOT, enzyme-linked immunospot; MVA, modified virus Ankara. Source: From Ref. 37.

lack of protective efficacy observed (41). A subsequent analysis of the environmental factors associated with loss of immunogenicity suggested association with a high prevalence of malaria parasitemia, but not helminth prevalence in these highly malaria-endemic villages (42). Work has also been done using different pre-erythrocytic antigens. The complete circumsporozoite protein (CSP) gene sequence from P. falciparum was cloned into both MVA and FP9 vectors and safety, immunogenicity, and protective efficacy has been evaluated. These vaccines were found to be safe and stimulate low to moderate levels of cellular immunity; however, no protective efficacy was seen against sporozoite challenge when these vaccines were used together in a heterologous prime-boost regimen (43), echoing the low immunogenicity and lack of efficacy of DNA-MVA vaccines encoding the circumsporozoite antigen (37). The U.S. Navy Medical Research group has investigated priming with a DNA vaccine expressing the P. falciparum CSP and boosting with recombinant protein with adjuvant (RTS, S/ AS02; see chap. 74), in clinical trials (44). The heterologous regime induced a detectable CD8þ T-cell response compared with no detectable CD8þ T-cell response when the recombinant protein was used alone. In this study, the immunogenicity of the DNA vaccine alone was not assessed, although it has previously been shown to induce some weak CD8þ T-cell responses when used alone (45,46). It is therefore not clear whether the CD8þ T-cell responses seen in the recent study were boosted by the recombinant protein or were simply a result of the DNA vaccination, and this study did not assess the protective efficacy of this regime. Recently the utility of a simian Ad, AdCh63, prime with an MVA boost regime has been assessed with the ME-TRAP

The current vaccine against tuberculosis (TB), BCG, is administered at birth throughout the developing world, and confers consistent, reliable and cost-effective protection against disseminated disease, particularly TB meningitis, in the first 10 years of life (47,48). However, protection against adult pulmonary disease is extremely variable, particularly in the developing world (49). Incorporating BCG into a prime-boost regimen would aim to allow the protective effects of BCG in childhood to be retained with the boost aiming to improve protection against adult pulmonary disease. It has recently been demonstrated in a large randomized controlled clinical trial in Brazil that homologous boosting of BCG with BCG did not improve protective efficacy against pulmonary disease (50). Several new TB vaccines, designed as heterologous BCG prime-subunit boost strategies, are among the most promising of these candidates that have entered into clinical trials. One such vaccine, MVA85A, is a recombinant MVA–expressing antigen 85A, a mycolyl transferase involved in mycobacterial cell wall synthesis. MVA85A was the first TB subunit vaccine to enter into clinical trials in September 2002 (51). A series of phase I clinical trials were conducted to evaluate the cellular immune responses induced with BCG alone, MVA85A alone, and BCG prime-MVA85A boost. The main immunological readout in these clinical trials was the ex vivo interferongamma (IFN-g) ELISPOT assay. The results of these early trials showed that a single vaccination with a relatively low dose MVA85A (5  107 pfu) in BCG naive, tuberculin skin test (TST) negative U.K. adults induced a highly significant and strong antigen-specific CD4þ T-cell response (29). These responses were considerably higher than had been seen previously using recombinant MVAs expressing other antigens. These results were attributed to the presence of preexisting central memory CD4þ T-cell responses induced by environmental mycobacteria, which may have ‘‘primed’’ a cellular immune response, which was then boosted by MVA85A (29). In the BCG prime-MVA85A boost trial, significantly higher immune responses were induced in the BCG prime-MVA85A boost group, when compared with either BCG or MVA85A alone, confirming that heterologous prime-boost immunization was more immunogenic than either vaccine alone (29) (Fig. 3). The optimal interval between priming and boosting vaccinations is an important consideration in the development of heterologous prime-boost immunization strategies. There are two periods during which one might wish to boost BCG vaccination: either in infancy, soon after BCG vaccination, or in adolescence, when the effects of BCG are starting to wane. It may also be desirable to boost at both time points. In the first BCG prime-MVA85A boost clinical trial, the median interval between BCG priming and MVA85A boosting was 18 years (modeling boosting in adolescence). A second clinical trial was conducted, where BCG naive, tuberculin skin test negative adults were recruited and vaccinated with BCG, and then

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Figure 3 IFN-g ELISPOT responses to antigen 85A peptide pools after vaccination with BCG alone, MVA85A alone, and BCG primeMVA85A boost [n ¼ 11(BCG); 14 (MVA85A); 17 (BCG prime-MVA85A boost]. Median interval between BCG prime and MVA85A boost was 18 years (29). *BCG-MVA85A responses significantly higher than BCG or MVA85A alone (p < 0.05). Abbreviations: BCG, Bacille CalmetteGue´rin; MVA, modified virus Ankara. Source: From Ref. 24.

boosted with MVA85A four weeks later (modeling boosting in infancy). The results of this second clinical trial were comparable to the previous clinical trial. Thus, at least using this immunological readout, it may not matter what the interval between BCG priming and MVA85A boosting is (52). The next clinical trial with MVA85A was conducted in subjects latently infected with M. tuberculosis, that is, M. tuberculosis itself was here acting as the ‘‘prime.’’ The safety and immunogenicity profile seen in this clinical trial was very similar to that seen in the BCG-primed subjects (53), so infection with M. tuberculosis may be as effective a prime for MVA85A boosting as BCG vaccination. There are now three other BCG-boosting vaccines in development, which have entered into early clinical testing. The first, Mtb72F, is a fusion protein of 32 and 39 kDa antigens from M. tuberculosis (54). This recombinant protein is being developed by GlaxoSmithKline Biologicals and is administered with the novel adjuvant, AS02A. This vaccine candidate entered into phase I studies in February 2004 (55). A second protein/adjuvant combination, ESAT6/85b, administered with a novel peptide/oligonucleotide-based adjuvant, IC31, entered clinical trials in November 2005 (55). A limitation of this vaccine is that one of the antigens, ESAT6, is a relatively M. tuberculosis–specific RD1 antigen utilized in two widely used new diagnostic tests (56). The inclusion of this antigen into any vaccine might confound such diagnostic tests, and it would be preferable to use other M. tuberculosis specific antigens in new vaccine candidates. A third BCG-boosting vaccine, a recombinant adenoviral vector (Ad35) expressing antigen 85A, B, and TB 10.4, entered into clinical trials in October 2006 (57). The first trial with this vaccine was conducted in BCG naive subjects in the United States, and a trial has now been completed in BCG-primed subjects in South Africa (Sadoff, personal communication).

HIV Within the field of HIV vaccines, several groups have explored the potential utility of heterologous prime-boost immunization strategies in enhancing cellular immune responses and efficacy in preclinical models. The candidates demonstrating promise in preclinical models have been assessed in early clinical trials, and three have advanced into efficacy trials to determine whether they protect against infection. There is also interest in the development of therapeutic vaccines for subjects already infected with HIV-1. A series of clinical trials using plasmid DNA as a prime and recombinant MVA to boost were undertaken in Oxford and Kenya. The vaccines used in these trials expressed the common immunogen, HIVA, which consists of consensus HIV-1 clade A Gag p24/p17 proteins fused to a string of clade A-derived epitopes recognized by cytotoxic T lymphocytes (CTLs) (58). These vaccines were found to be safe when used in heterologous prime-boost regimes (59,60). Weak and inconsistent CD4 T-cell immune responses to Gag were seen after DNA and MVA alone, but more reliable responses were seen after DNA prime-MVA boost, particularly when measured with the sensitive cultured ELISPOT assay (61). The dose of DNA (4 mg) and MVA (108 pfu) seemed to be critical and the T-cell responses after vaccination peaked at seven days after immunization and then diminished; other studies have also suggested the peak CD4þ T-cell immune response seen after recombinant MVA boosting is one week after vaccination (29,30). As in other studies, the bulk of the T-cell response was a CD4þ T-cell response; only a minority of donors gave CD8þ T-cell responses although when they occurred they were large and focused on single epitopes (61). These vaccines have also been evaluated in phase I studies in Kenya with similar results. It is worth noting that the same vaccines in mice and primates stimulated strong CD8þ (rather than CD4þ)

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T-cell responses, but this could have reflected the insertion of very strong immunodominant epitopes into the vaccine HIVA. A similar trial has been carried out with a DNA prime and NYVAC boost with HIV gag, pol, nef, and env immunogens. Tcell responses were detected using conventional ELISPOT assays in 90% of volunteers given the prime-boost regimen compared with only 37% given the NYVAC alone (62). Both CD4 and CD8 T-cell responses were seen to the envelope (Env). Weaker, mostly CD4, T-cell responses were seen to Gag and the other immunogens, that were similar in magnitude to the Gag-specific responses elicited by DNA-MVA (61). The HIVA-expressing DNA-HIVA and MVA-HIVA constructs expressing Gag, described above, have also been evaluated as therapeutic vaccines in subjects infected with HIV. These subjects were all on stable anti-retroviral regimens and had undetectable viral loads at the time of vaccination, although the virus is not eradicated and antigen must still be present (63). In these trials, DNA-HIVA and MVA-HIVA were administered alone and in prime-boost combinations. Interestingly, the MVAHIVA boosted both CD4þ and CD8 þ Gag-specific T-cell responses, which persisted for a year after vaccination (63). The CD8þ T-cell response was much larger. In this study, there was no difference between the immunogenicity of the DNA-HIVA prime-MVA-HIVA boost regime and MVA-HIVA alone trial (64). Interestingly, MVA-HIVA was markedly more immunogenic for CD8þ T cells in this HIV-infected group of subjects, in whom the prime is in fact HIV infection, than it was in HIV-negative subjects (61). These data, together with data from the TB trials described above, where BCG and environmental mycobacteria can act as the prime, suggests that in situations where the T-cell priming has been effective, a recombinant MVA is a very effective boosting agent. This implies that the challenge for prophylactic HIV vaccines and for vaccines against malaria is to identify the right prime, particularly if the aim is to elicit CD8 T-cell responses. An alternative approach, using a DNA vaccine prime expressing subtype B Gag-Pol-Nef fusion protein and modified Env constructs from subtypes A, B, and C and a recombinant adenoviral vector (human serotype 5) boost expressing the same immunogens is being developed at the Vaccine Research Centre (VRC) at the NIH. These vaccines have been evaluated alone in a series of phase I studies (24,65). These trials demonstrate immunogenicity of both vectors in all subjects, at least when administered at high dose. As expected, the recombinant adenoviral vector is more immunogenic than the DNA vaccine, when used alone. Interestingly both vaccines, when used alone, induce much higher CD8þ T-cell responses against Env than Gag, in contrast to natural infection where Gag responses are often greater than Env-specific responses. These VRC results differ from those reported at the 2005 and 2006 CROI Retrovirus Conference, where the Merck recombinant Ad5 vaccine that expresses p55 Gag, given alone, induced strong and durable Gag-specific CD8þ T-cell responses. One explanation for this difference is that in the Oxford and VRC studies Gag forms part of a fusion protein that may be unstable so that cross-priming is less efficient; studies in mice have demonstrated that stable protein antigens prime CD8þ T-cells more effectively (66). Thus, the Env is expressed intact and may therefore prime more effectively, though more recent data with separated Gag give similar results, suggesting intrinsic differences in processing and presentation of these antigens. A concern regarding Env as an immunogenic relates to its variability and the concern that vaccine-virus matching could be poor, possibly

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limiting the effectiveness of the vaccine, although there are some conserved regions in Env gp120 and gp41. In addition, large-scale studies in chronically infected African patients have shown that Gag-specific T-cell responses are associated with virus control, while Env specific T-cell responses are not (67). There is some argument, however, as to whether such results in chronic infection, where the immune system is chronically damaged, are relevant to prophylactic immunization. The Merck recombinant Ad5 vaccine, after modification to include three separate fusion constructs for Gag, Pol, and Nef, was taken forward into a large-scale efficacy trial. This phase IIb study, used recombinant Ad5 alone, in high HIV-risk people in North American and the Caribbean was aimed at showing that vaccination would result in reduced virus load after infection, as had been seen in macaques (68,69). Surprisingly the trial was halted in September 2007 because an interim analysis by the trial Data and Safety Monitoring Board showed no effect on virus load in vaccine recipients who became infected and evidence that the vaccine was enhancing the likelihood of HIV-1 infection. Further analysis revealed that the higher acquisition rates occurred in those who had preexisting antibodies to the vector, Ad5. Since the trial was stopped and unblinded and participants warned of possible dangers, monitoring of infections occurring in these volunteers continues so that it should be possible to determine whether the Adrelated risk is real or not. This will be important to know when considering the use of other Ad vectors in populations when there is an HIV risk. One plausible hypothesis for an increased risk is that Ad5 seropositivity correlates with the presence of memory CD4 T cells specific for the vector, that these possibly resided at mucosal sites and that these cells are in a state of activation that makes them prime targets for HIV-1. Whatever the explanation, it remains possible that the possible adverse effect in those who were immune to the vector masks a protective effect in those who were not. While this issue is being researched, the planned VRC trial of DNA prime with Ad5 boost, a combination that gave better results than Ad5 alone in a rigorous macaque-SIV challenge model, has been scaled down to a small trial only in people who are negative for antibodies to the Ad5 vector. In many parts of the world, the majority of the population is seropositive for Ad5 so this requirement would limit general application; however, other Ad vectors that have not previously infected humans remain possible candidates. The VRC trial will be an important test of the prime-boost concept with an efficacy read-out, which may complement prior data on immunogenicity (69). One (phase III) efficacy study is currently in progress, looking at a viral vector prime (ALVAC-HIV)—protein VaxGen gp120 B/E [AIDSVAX B/E in Thailand (ClinicalTrials.gov Identifier NCT00223080)]. This study is due for completion in June 2009 and it is noteworthy that the Data and Safety Monitoring Board has not prematurely stopped the trial because of adverse effects or futility. However, the approach has been controversial given that the ALVAC-HIV has previously shown poor T-cell priming and the gp120 protein antigen prime is unlikely to boost CD8þ T cells and has already failed to prime neutralizing antibodies (70).

Cancer Over the last decade, the application of heterologous primeboost technology has moved beyond infectious diseases into the field of solid tumor immunotherapy. A full review of this

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field is beyond the scope of this chapter. However there are some important cancer-specific issues that merit consideration here. Immunological tolerance needs to be broken for therapeutic vaccination against cancer to be effective. There are several ways in which cancerous cells evade immune recognition. MHC class I expression and tumor antigen expression can be reduced or lost, thus reducing T-cell recognition of a tumor cell. In addition, high levels of regulatory T cells are often present in cancer patients, which may inhibit or prevent any successful vaccination. The non-specific immunosuppression associated with cancer also contributes to the challenge of inducing sufficiently high levels of cellular immunity for therapeutic vaccination to work. The identification of key tumorassociated antigens to incorporate into potential therapeutic vaccines is a necessary first step. The enhanced cellular immunogenicity of heterologous prime-boost strategies discussed in this chapter may help overcome the immune evasion mechanisms by inducing a sufficient magnitude of cellular immune response to allow the immunosuppression and regulatory response to be overcome. The increasing number of potential cancer vaccines entering into clinical trials illustrates the potential of this strategy (71).

CONCLUSIONS AND FUTURE PROSPECTS Heterologous prime-boost immunization strategies are increasingly being evaluated in clinical trials and have been shown to be highly immunogenic. The lack of predefined immunological correlates of protection means that proof-of-concept efficacy studies are essential to see what level of immunogenicity is required for protection and whether improvements in immunogenicity translate into better efficacy. Studies in malaria, where small-scale efficacy trials are possible, and cancer patients where proof-of-concept in small numbers is also possible, are therefore important for comparative evaluations. Improvements in vector immunogenicity and safety will also potentially improve the applicability of this approach. Lessons learnt in one application will be potentially applicable to a wider range of indications.

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39 Mucosal Immunization and Needle-Free Injection Devices Jakub K. Simon and Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Bruce G. Weniger National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

Ana M. H. Restrepo Initiative for Vaccine Research, World Health Organization, Geneva, Switzerland

INTRODUCTION Currently, most vaccines are administered parenterally by needle and syringe. However, in industrialized countries, a proportion of children and adults have an aversion to injections. Therefore, administering vaccines without needles would be expected to increase compliance. Indeed, concern among parents about the increasing number of injections that must be given to infants and toddlers to deliver the recommended vaccines has been a strong impetus for the development of infant combination vaccines. The administration of vaccines by needle and syringe also poses occupational safety risks of needle sticks for health care providers. This could emerge as a more substantive issue in the future should it become necessary to immunize large populations expeditiously in response to a bioterror or emerging infection emergency. The role of needles and syringes in immunization practice is much more problematic in developing countries where major efforts are under way to increase immunization coverage and to introduce new vaccines. Except for the longstanding oral polio vaccine and the new rotavirus vaccines now used in some regions of the world, all the other vaccines recommended by the Expanded Program on Immunization (EPI) are given parenterally using needles and syringes. However, in many developing countries, injection safety is a notorious problem (1,2), as improper practices involving nonsterile needles and syringes (often used from one subject to another) cause abscesses and transmit blood-borne pathogens such as hepatitis B and C and HIV (2). Although parenteral vaccinations account for only a small fraction of all the parenteral injections given by health workers, immunization must be held to a higher standard because it involves healthy individuals. The Global Alliance for Vaccines and Immunisation (GAVI) and its associated Vaccine Fund are helping to strengthen immunization services in developing countries. GAVI supports the vision that immunization in the future would be more efficient, economical, and effective if all vaccines could be administered without the need for injection by needles. Three broad strategies are being advocated for needlefree administration of vaccines, each with its own advantages

(3). The first strategy involves mucosal immunization; the second is based on injection using needle-free devices; and the third involves transcutaneous immunization. Since an entire separate chapter of this book is devoted to transcutaneous immunization, this chapter will restrict itself to reviewing mucosal immunization and needle-free injection devices.

VACCINES DELIVERED VIA MUCOSAL SURFACES Potential and Practical Mucosal Sites for Administering Vaccines Although the human oral, nasal, rectal, conjunctival, and vaginal mucosa are all amenable to the application of vaccines for immunization, not all of these options are equally practical. For example, the rectal route, which has been shown to be highly efficient at eliciting immune responses in small studies in adults (4–6), might face cultural obstacles and be unpopular in some cultures globally; defecation by infants shortly after rectal immunization might also pose a problem that could diminish immunogenicity. Whereas antigens can be immunogenic after instillation into the conjunctival sac (7,8), some antigens might elicit conjunctival inflammation, which, on occasion, might lead to purulent conjunctivitis, secondarily. The vaginal mucosa, despite having relatively sparse inductive sites, can serve as a route for immunization against certain infections in females (9). Sublingual immunization can be considered a special form of oral immunization (10,11). Thus, in practical terms, oral and nasal administration are the most suitable options for all ages and both genders (12).

Inductive Sites for Immune Responses Aggregates of mucosa-associated lymphoid tissue are found along the mucosa of the gastrointestinal, respiratory, and genitourinary tracts. These include the gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue (BALT), and nasal-associated lymphoid tissue (NALT). Specialized microfold (M) cells overlying the mucosa-associated lymphoid tissues in the intestine and nose constitute competent portals by which antigens (including vaccines) can reach the

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underlying inductive sites for initiation of immune responses (13). These inductive sites are rich in antigen-presenting cells (dendritic cells, macrophages, etc.), in addition to the B and T lymphocytes that are present. Several reviews contain detailed descriptions of the anatomic architecture and cell types observed in the mucosa-associated lymphoid tissue (13–17). The gastrointestinal and upper respiratory mucosae are constantly exposed to antigens from resident normal bacterial flora of the gut and nasopharynx, as well as from food and inhaled materials. As a consequence, the mucosa of these anatomical sites can develop tolerance to an antigen so that a local immune response is either not elicited or muted. This is more likely to occur if the antigen is delivered in a soluble form and is given repetitively. Repetitive oral administration of a soluble antigen can also lead to systemic tolerance manifested by a diminished ability to mount an immune response upon parenteral administration of the same antigen (18). However, by administering the antigen of interest in a specialized delivery system or by coadministering strong adjuvants, potent immune mucosal and systemic responses can be elicited.

Immune Effector Responses Mucosally administered vaccines work well in preventing infections limited to the mucosal surfaces (e.g., cholera) (19,20) or those caused by pathogens that invade via the mucosa (e.g., poliovirus, Salmonella Typhi) (21–23). Dimeric secretory immunoglobulin A (SIgA) is by far the predominant Ig found in gastrointestinal and respiratory tract mucosal secretions, saliva and breast milk. Mucosal SIgA can mediate protection by neutralizing toxins, preventing virus entry into cells, neutralizing viruses within cells, or inhibiting the fimbriae and other virulence adhesins that bacteria utilize to attach to epithelial cell receptors. Because of the preponderance of SIgA in mucosal secretions, steps involved in the generation of this effector response have been the most extensively studied ones. Initial exposure of naive B lymphocytes to antigen in inductive sites in the mucosa-associated lymphoid sites (e.g., Peyer’s patches, NALT) and draining regional lymph nodes is followed by clonal expansion, isotype switching, affinity maturation, and migration (24,25). At approximately 7 to 10 days following administration of a mucosal vaccine, migrating cells can be detected as antibodysecreting cells (ASCs) found among peripheral blood mononuclear cells (26–33). Mucosal immunization elicits predominantly immunoglobulin (Ig)A ASCs that carry on their surface the a4b7 homing receptor that directs them back to the lamina propria in mucosal sites (34,35); they return mainly (but not exclusively) to the anatomic area from which they originated. The distribution of ASCs (mainly IgA) and memory B cells (36) accounts for both the compartmentalization of the mucosal immune system (SIgA responses are usually strongest at the site of induction) and the phenomenon of the ‘‘common mucosal immune system’’ (whereby SIgA responses can often be detected in mucosal sites remote from the inductive site) (37). There is reason to believe that if the relevant immune responses can be generated, systemic infections (including those that do not involve initial invasion from a mucosal surface, e.g., malaria) and toxicoses (e.g., tetanus) can also be prevented by administering the appropriate vaccines via mucosal surfaces (38). Indeed, properly formulated, mucosally administered vaccines can stimulate virtually any relevant type of immune response: serum IgG neutralizing antibodies

against toxins (38–40) and viruses (41–43) and the full array of cell-mediated immune responses including lymphocyte proliferation (44,45), cytokine production (44), and CD8þ cytotoxic lymphocyte activity (44,46–48), in addition to stimulating mucosal secretory IgA antibodies (29,49–54).

Duration of Protection Conferred by Mucosally Administered Vaccines It was previously taught that vaccines administered via mucosal surfaces can elicit only relatively short-term protection. However, there are multiple examples of mucosal vaccines that have conferred long-term protection and have elicited long-lived immune responses that correlate with protection. For example, an enteric-coated capsule formulation of Ty21a live oral typhoid vaccine conferred 62% protection over seven years of follow-up, and a more effective liquid formulation conferred 78% protection over five years of follow-up (55). A prototype nonliving oral cholera vaccine consisting of B subunit and inactivated whole vibrios conferred 56% protection over three years of follow-up (56). Titers of serum neutralizing antibody remain elevated for years following oral immunization with Sabin live oral polio vaccine (57).

Oral Vaccines Both live and nonliving antigens can be delivered orally, with good results. The trivalent attenuated Sabin poliovirus vaccine, the keystone of the global poliomyelitis eradication program, constitutes a paradigm that has encouraged the development and use of other live attenuated vaccines administered orally that have been licensed or are near licensure. The Sabin oral polio vaccine sets the standard for ease of administration to subjects of any age. Other licensed oral vaccines include Ty21a (Vivotif1) (55,58); live oral cholera vaccine strain CVD 103-HgR (previously commercialized under the trade names Orochol1 and Mutacol1) (59), whole-cell inactivated Vibrio cholerae O1 bacteria in combination with purified B subunit (Dukoral1), and inactivated V. cholerae O1 bacteria without B subunit (20,60). Overall, the experience with oral vaccines has been highly satisfactory. Nevertheless, problems have emerged. Postlicensure surveillance in the United States detected a rare association between tetravalent reassortant rhesus rotavirus vaccine (Rotashield1) and intestinal intussusception (61) that ultimately led to withdrawal of the vaccine from the market. Two new generation rotavirus vaccines (Rotateq1 and Rotarix1) were subsequently licensed following clinical development programs that included very large phase III field trials (*65,000 infants) that documented that risk of intussusception was not increased by vaccination (62,63). Postlicensure surveillance has corroborated the safety of these vaccines (64). Another problem that is being investigated is why some oral vaccines, particularly live ones, appear to be less immunogenic in individuals in developing countries compared with those in industrialized countries (65–70). Factors that can play a role include the presence of small bowel bacterial overgrowth (71), competing enteric viruses, or intestinal helminths (72). Various platform technologies are creating promising oral vaccine candidates. These include bacterial and viral live vectors expressing foreign antigens (38,73–76), DNA vaccines administered directly (77–79) or via bacterial vectors (80–83), transgenic plant ‘‘edible vaccines’’ (84,85), and various

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nonliving antigen delivery systems (liposomes, proteosomes, polylactide/polyglycolide microspheres, etc.), among others. Clinical trials with these technologies have generated mixed results (38,84,86), some of which have been promising (38,84).

Nasal Vaccines Nonliving and live antigens delivered nasally can also be highly immunogenic and protective. Indeed, in recent years considerable experience has been gained with an array of intranasal vaccines, recognizing that NALT is a particularly competent site for inducing immune responses. On the basis of its safety, immunogenicity, and efficacy (87,88), live coldadapted trivalent influenza vaccine (FluMist1), administered by a convenient single-use spray device that painlessly delivers large-droplet aerosol vaccine to the nasal mucosa, was licensed by the U.S. Food and Drug Administration and has proven highly effective in postlicensure assessments (89,90). The spray generated by the aerosol device (Accuspray1) (Fig. 1) produces large particles of a size that deposit on and remain confined to mucosa within the nose but do not generally descend to reach the bronchioles or alveoli. The Accuspray nasal spray device was used to deliver Edmonston-Zagreb (EZ) attenuated measles vaccine to adult volunteers with low titers of serum measles plaque reduction neutralizing (PRN) antibodies (Fig. 2). EZ delivered via nasal spray failed to significantly boost the subjects’ serum PRN titers (91) but did elicit anti-measles virus SIgA responses in nasal and oral washes (manuscript submitted). While EZ measles vaccine delivered intranasally by large-droplet aerosol spray did not elicit rises in serum PRN antibodies, small-particle (*3–5 mm) aerosol of EZ vaccine that allows particles to

Figure 1 Accuspray1, an intranasal drug delivery device manufactured by Becton, Dickinson and Company is used for the administration of cold-adapted influenza vaccine FluMist 1. Source: Courtesy of Becton, Dickinson and Company.

Figure 2 The Aerogen/Novartis device for delivering small-particle liquid aerosols that can reach the alveoli of the lung in infants and children. This device, as used by the World Health Organization project to deliver Edmonston-Zagreb measles vaccine by aerosol, includes a commercial vibrating mesh nebulizer modified with an inspiratory valve and expiratory filter. A multidose vial of reconstituted measles vaccine is fitted with a volumetric dropper, as used with oral polio vaccine. Two drops are dispensed directly on the vibrating mesh, and aerosol is administered for 20 seconds. Masks [silicon and disposable paper (inset)] are changed between subjects.

reach the alveoli has been immunogenic in multiple clinical trials (41,92,93), as described below. Various soluble and particulate nonliving antigens have been administered intranasally, with varying success. Research is seeking well-tolerated adjuvants and antigen delivery systems to enhance immunological responses to nonliving vaccines administered via the nasal mucosal surface. Cholera toxin (CT) and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are powerful adjuvants that enhance local SIgA and serum antibody responses to coadministered soluble or particulate antigens. Whereas these toxin adjuvants are unacceptable as human oral adjuvants (since as little as 5.0 mg causes severe diarrhea) (94), they have been investigated for nasal administration. Wild-type LT adjuvant was incorporated into a nasal nonliving influenza vaccine used in Europe (95). The vaccine was well tolerated and immunogenic in prelicensure clinical trials. However, postlicensure surveillance identified a likely association with cases of Bell’s palsy, resulting in withdrawal of that nasal influenza vaccine from the market. It is hypothesized that the promiscuous ganglioside binding mediated by the LT B subunits allowed toxin molecules to adsorb to facial nerve fibers and to translocate proximally, possibly resulting in neuronal damage (96). Efforts have been made to develop adjuvants that retain the potency and versatility of LT and CT for enhancing immune responses to coadministered antigens but with greatly improved safety profiles. One strategy has been to engineer mutant LT and CT molecules that exhibit reduced toxicity but

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retain adjuvanticity (97,98). Theoretical safety concerns remain to be resolved with these proteins because in some species the unaltered ganglioside binding properties of the mutant LT and CT molecules allow uptake by nasal olfactory nerve fibers and retrograde transport to the olfactory lobes of the brain (96). It is not known whether such neuronal transport of mutant LT and CT occurs in humans as well and, if so, what consequences might ensue. Accordingly, clinical trials using mutant toxins administered intranasally are carefully surveying for possible adverse effects. Safer intranasal adjuvants must be developed to realize more fully the potential of intranasal immunization with nonliving antigens. Several encouraging breakthroughs have been reported. One attractive adjuvant, referred to as CTA1-DD, links enzymatically active subunit A of CT to an Ig receptor binding peptide (99), thereby targeting the immune system’s B cells (99). One may envision that adjuvants like CTA1-DD could be coadministered intranasally with existing DTP, HBV, and Hib conjugate vaccines, as well as other relevant vaccines (e.g., pneumococcal and meningococcal conjugates or common protein vaccines), resulting in the stimulation of potent mucosal and systemic immune responses. Another promising approach involves use of the mucoadhesive polycationic calcium-containing polysaccharide ‘‘chitosan’’ as an antigen delivery system. Chitosan increases the transport of antigen across the nasal epithelium by altering intercellular tight junctions and decreasing the mucociliary clearance of antigen (100,101). In a phase I human clinical trial, diphtheria toxin cross-reacting material (CRM)197 given intranasally in a chitosan delivery system elicited significantly higher serum neutralizing antitoxin titers and SIgA antibodies than CRM197 administered without chitosan (40). These encouraging intranasal vaccine delivery strategies still have major hurdles to overcome. One of the most important will be to assess their effectiveness in immunizing infants in developing countries, recognizing that in such infants, upper respiratory infections and nasal discharge are highly prevalent (41).

Vaccines Delivered as Small-Particle Aerosols Liquid Aerosol Measles Virus as Vaccine Mortality owing to measles was reduced by 60%, from an estimated 873,000 deaths [95% confidence interval (CI), 634,000–1,140,000) in 1999 to 345,000 deaths (95% CI, 247,000– 458,000) in 2005 (102). Mass immunization campaigns in subSaharan Africa with parenteral measles vaccine administered by needle and syringe have decreased measles incidence and mortality by *90% where high immunization coverage has been achieved (103). Nevertheless, field experiences make it clear that such campaigns would be logistically simpler and safer if measles vaccine could be administered without needles (104–107). A significant number of clinical studies have been carried out with aerosolized measles vaccines starting in 1983 (108). Almost all of these studies have used the so-called ‘‘classical Mexican device’’ (CMD) and other variations on the Mexican model. Measles vaccine administered by aerosol (creating small particles that reach the lung) is safe, highly immunogenic, and efficacious (109–112). A recent systematic review reported that in children of age 10 months and older, aerosolized measles vaccine administration was more immunogenic than subcutaneous measles vaccine administration (93). The CMD generated highly encouraging clinical results but had

notable limitations in portability, robustness, practicality, and excessive inhalation time to vaccinate each subject. In 2002, the World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC), and the American Red Cross established the Measles Aerosol Project funded by the Bill and Melinda Gates Foundation to undertake the necessary preclinical studies and clinical trials required to achieve the licensure of a product (i.e., a device and vaccine) to administer measles vaccine by aerosol. In initial laboratory studies, WHO supported a detailed characterization of the aerosol generated by the CMD including the aerosol output and the aerosol size. The results for the CMD were aerosol output 0.34 to 0.40 mL/min and aerosol size of mass median aerodynamic diameter (MMAD) of 5 mm, with a geometric standard deviation (sg) of 1.87 (WHO unpublished data). Importantly, over a third of the droplets are 5 mm in diameter and are therefore able to reach the alveoli of the lungs (113,114). WHO invited almost two dozen aerosol device makers globally to participate in a detailed comparison of devices to identify three that would be suitable for further evaluation in phase I clinical trials. Each of these devices was assessed with respect to the character of the aerosol (i.e., aerosol output and aerosol size), retained vaccine potency (115), criteria that address practicality/field usability (size, portability, ease of use, power requirements, robustness), and cost per dose delivered. On the basis of results of this detailed comparison, three devices were selected for the phase I trial. Because of the lack of preclinical data performed under good laboratory practices (GLP), preclinical studies were deemed necessary to show the performance characteristics of nebulizers and the pharmacology and toxicology of aerosol measles. Safety studies in immunocompromised nonhuman primates given EZ measles vaccine by small-particle aerosol revealed no safety hazard compared with other routes of vaccination (116). Toxicology studies showed no local or systemic toxic effects related to the vaccine or placebo and provided additional evidence on the immunogenicity of this route of administration (117). Since the PRN titer is an accepted correlate of protection and a serological surrogate for efficacy, a standard operating procedure (SOP) was developed to standardize this important assay (118). The three devices were evaluated in phase I clinical trials with the EZ strain of attenuated measles vaccine manufactured by the Serum Institute of India. In this trial, the vaccine was administered to 145 healthy measles immune volunteers 1 to 35 years of age in three different sites in India. The measles aerosol vaccine was safe, well tolerated, and immunogenic (WHO unpublished data). A panel of experts was convened to conduct a thorough review of the available information regarding the three devices and identify the device(s) that was most suitable for use in immunization programs in developing countries. The decision was that the Aerogen/Nektar (now Aerogen/Novartis) device would proceed to the next phase trials. In 2009, WHO will initiate a phase II/III pivotal trial of the measles vaccine in healthy infants from 9 to 12 months of age who are eligible for their first measles vaccination. A total of 2000 infants will be enrolled in the study, randomly allocated 1:1 to the two arms (aerosol 1000, subcutaneous 1000). Nevertheless, important hurdles remain to be overcome with respect to aerosol administration. One is its safety in humans with immunodeficiency, including those infected with HIV. Although safety studies in immunocompromised

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nonhuman primates revealed no safety hazard compared with other routes of vaccination (116), safety in humans will need to be closely monitored because of interest in the use of the aerosol device in future mass vaccination campaigns in subSaharan Africa, including countries where HIV prevalence is high. Another issue is exposure of bystanders such as health workers and pregnant mothers holding their child receiving the measles aerosol. A concern exists for the transfer from one patient to the next of respiratory pathogens sneezed, coughed, or cried into the device as a result of the reuse of tubing or other parts of the aerosol pathway, and the economics and logistics of changing such parts between vaccinees. Another question is whether such disposables could be autodisabling to prevent inadvertent or intentional reuse between patients. The ongoing large-scale phase II/III clinical trial and related studies sponsored by the WHO are addressing some of these concerns. Dry Powder Aerosol Measles Vaccine Much attention is also being turned to the prospect of administering vaccines as dry powder aerosols that contain particles small enough to reach the alveoli. Once lyophilized measles vaccine is reconstituted to a liquid suspension, it is moderately heat labile and potency diminishes rapidly within hours. This is a limitation of delivery of measles vaccine as a liquid aerosol. Consequently, for many years, there has been interest in devices that can deliver measles vaccine (and other vaccines) as dry powders (119,120). Limited studies in nonhuman primates with dry powder measles vaccine formulations and delivery devices available at the time were not particularly encouraging (121). Dry Powder Aerosol Tuberculosis Vaccine Substantial preclinical progress has been made in exploring the delivery of dried Bacillus Calmette-Gue´rin (BCG) vaccine as small-particle and nanoparticle aerosols configured to reach the alveoli more readily (122,123). A key to this technology is the generation of ‘‘nanomicroparticles’’ of dried BCG vaccine that exhibit nanoscale dimensions in two axes but are micrometer in length. Particles manifesting this combination of nano- and micrometer-scale dimensions aerosolize more efficiently than spherical particles of similar diameter. Another feature of this technology is the use of BCG vaccine dried without a freezing step rather than lyophilized (i.e., freeze dried) BCG (122,123).

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was automatically replenished from the multidose vial following each injection. Beginning in the 1970s, studies raised concerns that MUNJIs could inadvertently transmit blood-borne infections, and in the mid-1980s, an outbreak of hepatitis B attributed to one device was well documented (128). Attempts to overcome this risk by fitting a MUNJI with a disposable cap through which the jet would pass but which would block the splashback of blood or serum to the nozzle failed to achieve its goal, as evidenced by the detection by polymerase chain reaction of HBV in 8% of subsequent ejectates following injection of HBV carrier volunteers (129). Consequently, health authorities discourage the use of MUNJIs, which have been replaced by a new generation of safer disposable-syringe jet injectors. The anthrax emergency in the United States in late 2001 and concern over the emergence of pandemic influenza viruses with high human transmissibility and virulence prompted public health authorities to contemplate how they might conduct mass immunization campaigns if their jurisdiction were confronted by a bioterror event or pandemic. Planning how limited numbers of health workers might administer vaccines rapidly en masse has reawakened interest in new highworkload jet injectors using cartridges that are easy to load and dispose of. If large clinical trials convincingly document their efficiency and safety, they could be utilized in mass campaigns in developing countries (e.g., against measles and meningitis) as well as industrialized countries (in pandemic situations or bioterror attacks). Simpler, single-dose jet injector devices are already in use for administering vaccines in lower-workload situations such as clinics for routine immunization. These devices also utilize autodisabling disposable cartridges and nozzles for individual patients to avoid cross-contamination (126). The Biojector1 2000 jet injector (Figs. 3 and 4) is currently utilized in some physicians’ offices and military clinics in industrialized countries to administer vaccines while avoiding needle stick injuries to health care workers (126,127). Although this device is not yet affordable for routine use in developing countries, newer, simpler, less-expensive devices have been developed for the market (e.g., PharmaJet1, LectraJet1, ZetaJet1).

NEEDLE-FREE INJECTION DEVICES Jet Injectors Jet injectors are needle-free devices that pressurize liquid vaccine to propel it through a small orifice (5–15 mm diameter) so as to pierce the integument to reach the skin, subcutaneous tissue, or muscle, as desired (3). In the half century of their use for vaccination since the 1950s, they have delivered millions of doses of many living and nonliving vaccines (124,125). Likely because of the passing through layers of skin where antigenpresenting cells are present, increased local reactions and also increased immune responses have been reported (126,127). From the 1950s to 1980s, high-workload multiuse nozzle jet injectors (MUNJIs) were widely utilized in mass immunization campaigns to deliver polio, influenza, smallpox, measles, yellow fever, and other vaccines (125). Multidose vials (containing up to 50 doses) permitted these devices to vaccinate 600 to 1000 subjects each hour using the same dose chamber, fluid path, and nozzle on consecutive subjects. The dosing chamber

Figure 3 Biojector1 2000, a high-pressure gas-driven needle-free jet injector that is licensed by the FDA for administration of vaccines intramuscularly or subcutaneously. By attaching this device to a large tank of CO2, it can be adapted for use in high-workload situations.

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Figure 5 LectraJet1 HS, a promising high-workload, ergonomic jet injection system with disposable, single-use vaccine cartridges (shown in a multiple-cartridge magazine). Source: Courtesy of D’Antonio Consultants International, Inc.

Figure 4 Wheal in the skin of an adult volunteer immunized with a measles DNA vaccine administered intradermally using the Biojector1 2000 needle-free injection device.

One drawback of the current devices is that their cartridges or syringes are not prefilled by vaccine manufacturers. The end users must transfer the dose from vaccine vial into the cartridge by means of a special vial adapter, an extra step comparable with filling a conventional syringe manually (130). Such filling steps could be entirely eliminated if manufacturers prefilled vaccines directly into jet injector cartridges, ideally a common one that could fit a variety of injectors. One such system, since abandoned by its manufacturer, was the Imule1 cartridge (131), which was used in a manually cocked, springpowered investigational injector called the Mini-Imojet1 (131). It gave promising results in pediatric and adult clinical vaccine trials in both industrialized and developing countries (131). A needle-free jet injector with a promising design that permits fast and finger-free loading and unloading of cartridges is the LectraJet, which comes in both a high-speed (HS) model (Fig. 5) and a low-workload configuration M3 model. The HS system has been designed for a large-scale, high-workload scenario of mass campaigns. Its individual autodisabling syringe can be filled on-site or prefilled by the vaccine manufacturer. To maximize speed, syringes are held and extracted from a ‘‘magazine,’’ which can be mounted on a table, other fixed object, or even the vaccinator’s other forearm. After the injection, the used cartridge is discarded into a trash receptacle with the push of a button, and the injector is ready to reload by pushing against another cartridge in the magazine. This ‘‘hands-free’’ process allows for as many as 600 injections per hour. The LectraJet HS is electrically powered by means of rechargeable batteries or via an electrical outlet. For use when electrical power may not be reliable, the manually cocked LectraJet M3 model can be used (Fig. 6). The LectraJet system is still investigational, but licensure in the United States is expected in 2009.

Figure 6 LectraJet1 M3, a manual jet injector that utilizes the same cartridges as the LectraJet HS but can be powered by manual or pedal cocking devices and does not require electricity. Source: Courtesy of D’Antonio Consultants International, Inc.

One advantage of jet injectors over some other needlefree devices for vaccination is that they are capable of using existing, off-the-shelf vaccines, whose delivery by this route has been well documented for a variety of protein and polysaccharides subunit vaccines, combination vaccines, and

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live attenuated viral vaccines. They have also shown promise for successfully delivering DNA vaccines in clinical trials (132–134).

SUMMARY COMMENT Advances in mucosal immunization, improved needle-free injection devices, and the rapidly developing field of cutaneous immunization all engender optimism that vaccines will be increasingly administered without the need for needles. Since each of these strategies has its own particular advantages, it is likely that all three will be needed to realize the ultimate vision when all immunizations in all age groups can be administered without needles.

DISCLAIMER Please note that trade names of commercial products are mentioned for identification purposes only, and do not constitute endorsement nor imply recommendations for their use by the authors or their affiliated institutions.

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127. Jackson LA, Austin G, Chen RT, et al. Safety and immunogenicity of varying dosages of trivalent inactivated influenza vaccine administered by needle-free jet injectors. Vaccine 2001; 19(32): 4703–4709. 128. Canter J, Mackey K, Good LS, et al. An outbreak of hepatitis B associated with jet injections in a weight reduction clinic. Arch Intern Med 1990; 150(9):1923–1927. 129. Kelly K, Loskutov A, Zehrung D, et al. Preventing contamination between injections with multiple-use nozzle needle-free injectors: a safety trial. Vaccine 2008; 26(10):1344–1352. 130. Jodar L, Duclos P, Milstien JB, et al. Ensuring vaccine safety in immunization programmes–a WHO perspective. Vaccine 2001; 19(13–14):1594–1605. 131. Parent du Chaˆtelet I, Lang J, Schlumberger M, et al. Clinical immunogenicity and tolerance studies of liquid vaccines delivered by jet-injector and a new single-use cartridge (Imule): comparison with standard syringe injection. Imule Investigators Group. Vaccine 1997; 15(4):449–458. 132. Roy MJ, Wu MS, Barr LJ, et al. Induction of antigen-specific CD8þ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine. Vaccine 2000; 19(7–8):764–778. 133. Tavel JA, Martin JE, Kelly GG, et al. Safety and immunogenicity of a Gag-Pol candidate HIV-1 DNA vaccine administered by a needle-free device in HIV-1-seronegative subjects. J Acquir Immune Defic Syndr 2007; 44(5):601–605. 134. Graham BS, Koup RA, Roederer M, et al. Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine. J Infect Dis 2006; 194(12):1650–1660.

40 Advances in Transcutaneous Vaccine Delivery Robert C. Seid, Jr. and Gregory M. Glenn Intercell USA, Inc., Gaithersburg, Maryland, U.S.A.

INTRODUCTION Transcutaneous immunization (TCI) is a noninvasive, painfree, and easy-to-use vaccination technique that introduces antigens and/or adjuvants topically onto the skin in the form of a patch, a liquid solution, or ointment. The skin is one of the largest immune organs and forms an integral part of an immune system network known as the skin-associated lymphoid tissue (SALT) (1). The skin epidermis is naturally populated with numerous resident professional antigen-presenting cells (APCs) known as Langerhans cells (LCs). These APCs form an immune network to efficiently capture microbial pathogens or antigens that have penetrated through the skin’s outer barrier, the stratum corneum (SC), and have entered into the epidermal tissues. LCs play an important role in the induction of immune responses. APCs sample and process pathogen-derived antigens and traffic to nearby regional skin, draining lymph nodes (DLN) to present the antigen fragments to naı¨ve resting T and B cells to induce production of antigenspecific CD4þ and CD8þ T cells as well as systemic and mucosal immunity (2–5). Because the SALT system is capable of mounting effective immune responses to pathogen-derived antigens (1), the skin has become an attractive, noninvasive route for vaccine delivery. TCI targets delivery of antigens through the SC and into the epidermis to exploit the skin’s immune system. Within the last decade, remarkable progress, as evidenced by >200 publications, has been made in developing TCI as an alternative vaccine delivery route. Based on many of these published studies, some general principles regarding the TCI approach have emerged. First, there appears to be no restriction to the type of antigens or their molecular size that can be used for TCI. Successful skin immunizations have been conducted with a variety of protein and DNA antigens, including those derived from bacterial and viral microorganisms. Molecular sizes for TCI have ranged from small to medium-sized peptides carrying T-cell and cytotoxic T lymphocyte (CTL) epitopes (6–9), to large molecular weight subunit protein antigens (10–13), to split and whole-inactivated viruses (14–16). Second, minor disruption of the SC at the site of vaccine application enhances efficiency of antigen penetration and skin immunity. SC disruption can be achieved by physical or chemical means and is further discussed in this chapter. Third, adjuvants can be used to generate a robust immune response to coadministered antigens by TCI. Adjuvants commonly used are cholera toxin (CT) from Vibrio cholerae and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). These potent adjuvants are too toxic to be administered orally or intranasally, but can be safely used on the skin (3,4,15).

In this chapter, we present recent insights and strategies in using TCI as a new vaccine delivery paradigm. Topics discussed include (i) skin structure as related to immunology; (ii) methods for SC disruption, including Intercell’s skin preparation system (SPS) device; (iii) types of skin adjuvants used; (iv) recently published TCI applications; and (v) Intercell’s dry patch formulation technology for preparing thermostable vaccine patches for LT and influenza. Finally, we describe Intercell’s recent clinical studies using a dry LT-patch for the prevention and reduction of travelers’ diarrheal illness, and as an immunostimulant (IS) patch for a pandemic influenza vaccine candidate.

SKIN STRUCTURE AND IMMUNE FUNCTION The skin consists of three layers—the SC, epidermis, and dermis. In humans, the SC is approximately 10 to 20 mm thick and is composed of dead keratinocytes cells surrounded by a lipid mortar. The epidermis, which underlies the SC, is a continuously growing layer of epithelium (50–100 mm) that consists of about 90% to 95% of keratinocytes at various progressive stages of differentiation. The remaining 2% to 8% of the epidermal cell population consists of immature dendritic cells, known as LCs. Because of their dense population and long dendritic protrusions, LCs form a network that covers about 20% of the entire surface area of the skin. The dermis (1–3 mm thick) supports the epidermis with connective tissue and contains blood vessels, lymphatics, nerve endings, hair follicles, and sweat glands. The dermis also contains dendritic cells and mature LCs in transit, but the density of these APCs in the dermis does not match that of the epidermis (2,3,15). Vaccine delivery by TCI is targeted to the superficial layer of the epidermis due to the presence of the immature LCs, which are sufficiently networked to sample and process microbial antigens. In their normal resting state, the immature LCs express low levels of cell surface histocompatibility complex (MHC) molecules and costimulatory molecules. This resting state is influenced by constitutive secretion of IL-10 and transforming growth factor TGF-b cytokines by the neighboring keratinocytes (17). Upon encountering a microbial ‘‘danger signal,’’ the LCs become activated, triggering uptake and processing of the antigens that results in LC migration from the skin epidermis via afferent lymphatics to the DLNs, where they present the processed antigens to both CD4þ and CD8þ T lymphocytes to initiate antibody and cellular immune responses. During their migration to the DLN, the LCs undergo a maturation process that includes upregulation of membraneassociated co-stimulatory and adhesion molecules (i.e., CD80,

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CD86, ICAM-1), transport of MHC-peptide complexes to the cell surface, increased expression of CCR7 chemokine receptors that triggers the migration of LCs, and secretion of cytokines, such as IL-12 and IL-1 to influence T-cell differentiation into effector and memory T-cells to enhance adaptive immune resistance to microbial products (17–19). The immunocompetent keratinocytes, which are the most abundant cells of the skin epidermis, also play an active role in initiating innate and adaptive immune responses. Keratinocytes express a wide range of Toll-like receptors (TLRs) on their cell surfaces (20,21). When exposed to a danger signal of a microbial pathogen or component, the keratinocytes secrete proinflammatory cytokines, which can modulate the antigen processing and presentation by the LCs, as well as stimulate their migration to the DLNs. In addition, the inflammatory cytokines promote migration of skin-recruited macrophages, dermal dendritic cells, and neutrophils to the DLN for participation in T-cell priming. Skin vaccination can offer certain advantages over intramuscular (IM) immunization. First, the skin immune system can generate antibody responses, often at reduced antigen doses. By coming into contact with a dense population of APCs, a smaller antigen dose can generate an effective, robust immune response. For example, vaccination with one-tenth of the typical dose of live attenuated smallpox vaccine by skin scarification (20,22), or with one-fifth the dose of seasonal influenza (23), is feasible in humans. The reduced dosage takes advantage of the effectiveness of the skin immune system in generating an immune response to the viral vaccine. In addition, smaller antigen dose requirements (i.e., dose-sparing) have been demonstrated through skin immunizations in a number of animal studies, using the TCI approach with adjuvants. In these experiments, the adjuvant was either coadministered with the antigen or used as an immunostimulatory patch that was placed over the vaccine injection site (15,24–27). Second, the skin immunization can generate mucosal responses, both IgG and IgA, at multiple sites including the oral and nasal cavity, gut, lung, saliva, and female reproductive tract (2,28). Parenteral immunization induces specific humoral antibodies, but often falls short in generating specific mucosal immune responses. Third, skin immunization can induce antigen-specific CD4þ(T-helper) or cytotoxic CD8þ T-cell responses to topically administered peptides that mimic epitopes from the antigens and whole proteins (6–9). Synthetic peptides are not generally effective immunogens when given via parenteral injection. Moreover, parenteral immunizations with non-replicating vaccines such as protein antigens induce mainly humoral antibody responses, and do not typically induce strong cellular immunity. A recent TCI study demonstrated that the activated LCs carrying an HIV peptide antigen delivered by the skin migrated primarily to the DLN, but were shown to migrate from the skin epidermis to the gut mucosa where they induced a CTL immune response to the delivered peptide antigen (7). Thus, the advantages offered by skin immunization compare favorably to the normal practice of parenteral immunization by needle, which perforates the skin and bypasses the SALT system that should be of most interest to vaccinologists.

METHODS FOR SKIN DISRUPTION AND INTERCELL’S SPS SYSTEM The outermost layer of the skin, the SC, provides a natural skin barrier against water loss and various external physical, chemical, and mechanical stimuli. It also represents the major

obstacle to vaccine delivery via the transcutaneous route. To improve vaccine delivery into the skin epidermis, significant research efforts have been focused on methods to gently remove, disrupt, or even modify the structure of the SC barrier. Common mechanical disruption techniques such as tape stripping with D-Squame or 3M tape, or gentle skin abrasion with emery paper or pumice swabs have been used to remove or disrupt the SC to facilitate antigen delivery into the skin (2,28). Hydration of the SC with occlusive backing has been used to promote swelling of the keratinocytes and accumulation of fluids in the intercellular spaces, thus allowing antigens to pass through the skin easily (2,28,29). The anionic surfactant, sodium lauryl sulfate, has recently been used to disorganize the SC by extracting lamellar lipids and enhance the skin’s permeability for vaccination (30). Other innovative physical methodologies to facilitate deposition of antigens into the skin epidermis include (i) microneedle arrays, which employ shallow antigen-coated silicon projections to penetrate into the epidermis (31–33); (ii) ballistic particle-based guns that deliver vaccine powders to the superficial layer of the skin (34–36); (iii) low-frequency ultrasound devices to increase skin permeability through disruption of the SC by acoustic cavitation (37); (iv) microporation devices, which use thermal energy to vaporize small areas of the SC (38); and (v) electroporation devices, which use short electrical pulses to create transient pores in the lipid bilayer of the SC (39,40). Most of these energy-powered devices have been tested and evaluated only on rodent skins. Further development of these physical devices is required to evaluate their safety in humans. Moreover, since some of these devices are large in size, expensive, and require electrical power, their uses will be suboptimal for routine immunization and mass vaccination campaigns. Intercell has recently developed a cost-effective skin preparation device to mildly disrupt the SC to enhance vaccine delivery. The device, named the SPS, has been engineered to be simple to use, well tolerated, disposable, and readily adaptable to high volume manufacturing techniques (15,41). As discussed below, use of the SPS results in highly consistent, high levels of seroconversion in response to TCI immunization via a patch. The SPS is a small, handheld device with a platform called the mask that contains an aperture over which an abrasive strip is pulled (Fig. 1A). A small push-button over the aperture contains a force control dome that provides controlled pressure to allow contact with the abrasive surface and the skin. The strip is pulled out, bringing the abrasive surface on the strip over the skin. The SPS is composed of standard materials, including a commercially available medical tape and medical-grade plastics. Materials and manufacturing processes were selected to enable low-cost, high-volume manufacturing of a commercial version of the present device. The force control button provides audible and tactile feedback to the user when the designated force is reached. An abrasive strip of EKG-style, fine grit sandpaper is used to provide mild SC disruption. The mask serves several roles: providing a controlled surface over which the strip is pulled; controlling the treated surface via the aperture; and masking the sensation of the abrasion when contact is made with the skin. In an adult volunteer study, subjects were provided with a prototype SPS and were allowed to pretreat themselves on the thigh by following graphically illustrated instructions. After self-pretreatment, a dry patch containing E. coli heat-labile LT

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Figure 1 Intercell’s skin preparation device and dry patch system. (A). In this overhead photo, Intercell’s single-use disposable skin preparation device is shown relative to the size of a U.S. quarter. (B). shows a schematic diagram of the patch components.

was placed at the pretreatment site. A separate group was also pretreated on the thigh by clinicians using the prototype SPS followed by placement of the dry LT patch. Two doses of 50 mg of LT were used and anti-LT antibody responses were then measured. No difference was seen in the physician versus selfpretreated group (24.5- vs. 26.8-fold rise in the serum IgG), and 95% seroconversion was achieved in both groups (15,41). The SPS has been specifically designed for simple use, including self-application, and these data suggest that a self-applied vaccine patch system can be developed.

ADJUVANTS FOR SKIN IMMUNIZATION The presence of an adjuvant is critical to induce a strong systemic and mucosal immune response to the coadministered antigen by TCI. Various adjuvants have been used for skin immunization, and since they can bias immunity toward a Th1 or Th2 immune response, their selection should be based on whether a Th1 or Th2, or mixed Th1/Th2, immune response is preferred. Furthermore, skin immunization allows the use of potent adjuvants, whereas their use would be prohibited for parenteral or mucosal administration because of the toxicity they exert.

Besides being strong immunogens, the bacterial ADP-ribosylating exotoxins (bAREs), such as E. coli LT and CT, are potent adjuvants in the context of the skin. Numerous preclinical and clinical studies (3,11,15,24) have documented the safe use of bAREs on the skin. Our laboratory has safely applied the skin patch containing LT in over 3,000 human subjects without any vaccine-related serious adverse events (SAEs), and the LT patch is currently under U.S. IND for phase II clinical trials. As potent immunogens with strong adjuvant properties, LT and CT are commonly used in TCI protocols to induce systemic and mucosal antibodies against the toxins themselves, as well as to various types of coadministered vaccine antigens including proteins, peptide antigens, and whole and split viruses. Importantly, preexisting antibodies to LT or CT do not adversely affect the adjuvanticity of these adjuvants on repeated TCI administrations. In this respect, TCI is akin to intranasal (IN) or oral mucosal immunization with adjuvants, as the simple admixture of LT with a coadministered antigen such as tetanus toxoid (TTx) or influenza hemagglutinin (HA) on the skin results in markedly higher systemic and mucosal antibody responses compared to the administration of antigens alone. The basis for the adjuvanticity of bAREs has been described to some degree. Observations that the enzymatically active holotoxins are more potent immunoadjuvants in inducing systemic antibody responses, while their purified B-subunits are marginally effective, support the notion that the strong adjuvant action is associated with their ADP-ribosylating enzymatic activity (11,15). In support of this hypothesis, a number of molecular events have been proposed to explain the strong adjuvant action of bAREs on the skin. The LT (or CT) holotoxin consists of homopentamers of B subunits (LTB or CTB) associated with a single A subunit (LTA or CTA). The LTB binds specifically to GM1 ganglioside receptors that are expressed on skin epithelial cells, that is, the LCs and keratinocytes. The LTB/GM1 binding facilitates the entry of LTA into the cells. The LTA enzymatically ADP-ribosylates the Gs protein of adenylyl cyclase, which results in increased levels of cAMP. The rise in cAMP signals the skin epidermal cells (keratinocytes and LCs) to stimulate the secretion of proinflammatory cytokines such as IL-1 and TNFa, which in turn act on LCs to trigger their maturation and migration to regional lymph nodes, as well as to other distal mucosal sites, where they present peptide fragments to naı¨ve T-cells. Some have suggested that binding to the GM1 receptors is more critical than ADP-ribosylating activity in explaining the adjuvant effect of bAREs. For instance, the binding of LT or CT through their B subunit to ganglioside receptors on skin epithelial cells can induce changes in membrane channel potential that results in altered skin permeability and enhanced penetration and uptake of coadministered antigens in mice (42). However, this explanation is inconsistent with human skin biopsies, which had no microscopic changes in spite of the induction of robust immune response via TCI (5). Another group has suggested that the adjuvant effect is due to the bAREs’ ability to disrupt the skin barrier function by inducing apoptosis of the skin keratinocytes. This apoptosis of the skin keratinocytes, in turn, creates intercellular spaces or voids in the skin that allow more efficient and rapid diffusion of adjuvant and antigen molecules to the underlying epidermis, where they are taken up and processed by the LCs (17,43).

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As skin adjuvants, LT and CT, appear to give similar enhanced immune responses to coadministered soluble antigens (11). The antibody response induced by LT and CT to the coadministered antigen is predominantly IgG1, characteristic of the Th2 response. It has been reported that LT can induce a stronger Th1 response than CT, and a mixed Th1- and Th2-immune response has been observed. CT, on the other hand, is thought to provoke predominantly a Th2 response, which is characterized by production of IgG1 and IgA antibodies, and CD4þ T-cells producing IL4, IL5, and IL10.

TCI Applications for Bacterial Diseases Haemophilus Influenzae TCI has also been used to deliver a complex bacterial product, such as a semi-synthetic glycoconjugate vaccine. Mawas et al. (49) delivered a Haemophilus influenzae type b (Hib) glycoconjugate vaccine coadministered with CT or LT mutants onto rats’ skin. The glycoconjugate elicited high antibody titers to the capsular polysaccharide of Hib and to the protein carrier. The anti-Hib polysaccharide antibodies were shown to be passively protective in an infant rat model against a virulent strain of Hib.

CpG Motifs TLR agonists have been evaluated as potential adjuvants to enhance the adaptive immune response. For instance, unmethylated bacterial CpG motifs or synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) are readily recognized as a danger signal by the TLR9 expressed by dendritic cells, macrophages, monocytes, and splenocytes (44). Ligation of CpG to TLR9 triggers the induction of cellsignaling pathways that cause production and release of proinflammatory Th-1 driving cytokines such as IL-12, TNFa, IL1a, and IFN-g. Thus, the CpG motifs can have a broad adjuvant effect on a coadministered antigen, and skew the immune response to a Th1 phenotype. In a TCI study, a synthetic CpG-ODN was used in combination with CT to modulate the immune response of a synthetic peptide representing a T-helper epitope of influenza virus HA (6). The CpG-ODN acted synergistically with CT to increase the proliferative T-cell responses to the HA peptide. Thus, the bias toward Th2-type responses stimulated by CT was shifted toward Th1 phenotype because of the presence of CpG in the HA peptide/CT formulation. Interestingly, the CpG administered without CT on the skin exhibited a weak adjuvant effect to the HA peptide, thus highlighting the potent immunomodulatory properties of CpG in combination with ADPribosylating exotoxins for TCI application. This and other studies (6,12,45,46) showed that CpG administered with CT or LT can enhance TCI-induced immune responses, and can modulate a Th2 to a Th1 type response.

Imiquimod and Other Adjuvants Used on Skin Imiquimod, which has been used in topical treatment of genital warts, is a potential adjuvant for TCI (43,47,48). This small molecule has been shown to bind to TLR7 receptors expressed on several dendritic cell (DC) subsets, including skin LCs. The binding gives rise to production of inflammatory cytokines, such as IL-1, IL-6, IL-8, TNF-a, and IFN-a, some of which are critical for antigen uptake by LCs and their migration into the DLNs. Studies have shown that imiquimod can be transcutaneously applied with a peptide antigen to mount a CTL response that is specific for the epitope used for immunization. Interestingly, transcutaneous peptide immunization with imiquimod as the adjuvant does not seem to require disruption of the skin barrier (47).

EXAMPLES OF RECENT TCI APPLICATIONS TCI has been demonstrated to elicit humoral and cellular immune responses against a wide range of bacterial and viral antigens. Some recent TCI studies illustrating the use of the skin as a noninvasive route for administering antigens are described below.

Bacillus Anthracis In a previous TCI study in mice, a recombinant protective antigen (rPA) of B. anthracis coadministered with LT on the skin was shown to induce long-lasting neutralizing antibody titers that were superior to those obtained by IM injection of alum-absorbed rPA (50). Moreover, the TCI-induced anti-rPA antibodies completely protected TCI-immunized mice against challenge with Bacillus spores from an unencapsulated strain. In a more recent TCI study, Peachman et al. clearly demonstrated the superiority of TCI over the injected alum-adsorbed vaccine in protecting vaccinated mice against intranasally administered Bacillus spores from a more virulent encapsulated strain (51). A significant correlation was observed between the TCI-induced toxin-neutralizing antibody titer and mouse survival after the intranasal challenge. Chlamydia An ideal vaccine for Chlamydia trachomatis should induce (i) mucosal IgG and IgA to prevent infection by Chlamydia elementary bodies and (ii) a strong cell mediated immune response to limit ascending infection to the uterus and fallopian tubes. Berry et al. (52) have shown in a mouse model that TCI with Chlamydia major outer membrane protein (MOMP) in combination with both CT and CpG can elicit MOMP-specific IgG and IgA in vaginal and uterine lavages, MOMP-specific IgG in serum, and IFN-g-secreting Th1 cells in the reproductive tract lymph nodes. More importantly, the TCI protocol enhanced clearance of Chlamydia organisms following vaginal challenge. Cholera The toxin-coregulated pilin A (TcpA) is a second major virulence factor of V. cholerae and is essential for colonization in animal models and humans. Rollenhagen et al. (53) have immunized mice transcutaneously with TcpA with CT, and the immune responses elicited were protective in a mouse cholera challenge model. Interestingly, TCI application of TcpA with CT did not induce anti-TcpA serum IgA, despite induction of prominent anti-TcpA IgG responses. Three TCI applications of TcpA and CT were required to induce the protective anti-TcpA responses in mice, while TCI applications of TcpA without CT did not develop anti-TcpA responses. Clostridium Clostridium difficile, the leading cause of nosocomial diarrhea in the industrialized world, causes more than 300,000 cases of diarrhea in the United States and can lead to colitis, toxic megacolon, systemic inflammatory response syndrome, and

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death. Currently, no C. difficile vaccine is commercially available, although a parenteral vaccine candidate consisting of formalin-detoxified C. difficile toxins A (CDA) and B (CDB) has been evaluated in phase I (54,55). The current thought is that anti-toxin A protects better than anti-toxin B, and that systemic and mucosal responses are required for protection. Recently, Ghose et al. (56) found that TCI with CDA  CT induces anti-CDA IgG and IgA responses in serum and antiCDA IgA responses in stool. Sera from TCI-immunized mice were able to neutralize CDA activity. Parenteral immunization with CDA alone (no CT) did not induce serum or stool antiCDA responses despite repeated immunizations. Plague The recombinant F1 (rF1) and V (rV) proteins from Y. pestis are two promising vaccine candidates against plaque (57). When mice are injected with formulations containing rF1 and rV together with alum, they can be fully protected against injected or aerosolized plague challenge (57). Recently, Eyles et al. have shown that two or more TCI applications of rF1 and rV admixed with CT could induced significant increases in serum anti-F and anti-V antibodies in mice, and that three TCI applications conferred full protection against challenge with a virulent strain of Y. pestis (58). Also, splenocytes from a single TCI immunization were shown to secrete significant quantities of IL-6, indicating a cell-mediated immune response; however, low numbers of F1/V-specific antibody-forming cells in the spleens were observed. The investigators concluded that TCI immunization with F1 and V antigens may require higher doses and more frequent administration to match the levels obtained by the other routes of administration, such as IN and intradermal (ID) delivery. Their study also showed that TCI with the plague antigens and CT were effective for priming responses that could be boosted by the IN or ID routes. In their TCI study, the mouse skin was shaved and wetted with damp cotton wool prior to application of the antigens and adjuvant. It is possible that a stronger immune response could have been elicited if another skin pretreatment method was used.

TCI Applications for Viral Diseases Herpes Simplex El-Ghorr et al. (59) have applied whole inactivated herpes simplex type-1 virus (HSVi), as well as HSV-1 antigens (HSVag), with CT as adjuvant onto mice skins. Both HSV preparations by TCI resulted in the production of serum and mucosal (fecal) antibodies to HSV. Surprisingly, their study showed that HSVi  CT was a more potent stimulator of humoral immunity, while HSVag  CT was the more potent stimulator of cell-mediated immunity. In a mouse epidermal challenge model, HSVag  CT vaccine gave a higher level of protection than the HSVi  CT vaccine, a result suggesting that the efficacy of HSV vaccines is more dependent on cell-mediated, rather than humoral immune responses. Respiratory Syncytial Virus Currently, there is no effective vaccine against respiratory syncytial virus (RSV), which is the leading cause of severe lower respiratory infections in newborns and young infants. A formalin-inactivated alum-adsorbed RSV vaccine was tried in the 1960s in young infants. It failed to protect, and resulted in exacerbated disease when natural RSV infection occurred (60). Godefroy et al. (61) have recently investigated the TCI

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applications of two G protein-derived molecules, G2Na and G5, in mice. The former protein contains T- and B-cell epitopes, and the latter is a pure B-cell epitope. Three topical applications with G2Na  CT were shown to elicit antigen and adjuvant specific antibody responses. In contrast, G5 was not immunogenic when given topically with CT, suggesting that coupling of G5 with T-helper sequences may be necessary. In correlation with the anti-specific antibody titers, TCI with G2Na  CT significantly protected mouse lung tissue against RSV infection and reduced RSV infection in the nasal tract, while G5  CT did not. The investigators concluded that an RSV vaccine delivered transcutaneously is a viable concept, and could be tested in clinical trials. Seasonal Influenza Skountzou et al. (16) have investigated the potential of TCI using formalin-inactivated whole influenza virus that exists as large particulate antigens with molecular weights around 250 MDa. TCI applications of inactivated influenza particles on mouse skin induced virus-specific anti-influenza antibodies with hemagglutination inhibition and neutralizing activities, and conferred protective immunity to virus challenge. Coadministration with CT was found to significantly enhance the humoral and cell-mediated immune responses against the influenza antigen. Moreover, the investigators found that pretreatment of the mouse skin with oleic acid (OA) and retinoic acid (RA) significantly enhanced immunogenicity and conferred enhanced protection. Their data suggested that these two known penetration enhancers may facilitate the skin penetration of influenza virus particles to allow more frequent interactions with LCs, or to enhance the migration of the antigen-loaded LC to the DLNs, or to directly affect immune cells since both OA and RA can induce secretion of IL-10. As described further in this chapter (sect. ‘‘Trivalent Inactivated Split-Influenza Patch), our laboratory has developed a dry thermostable patch formulation containing a trivalent inactivated split-influenza vaccine (TIV), and demonstrated its effectiveness in inducing anti-influenza antibodies in a guinea pig immunogenicity model (14). When administered with LT, the dry TIV patch induced a robust immune response that was comparable to or better than the response to an injected TIV vaccine. HIV Because it plays a critical role in virus replication and disease progression, the HIV-1 Tat protein represents a promising vaccine candidate against HIV infection. Using a synthetically prepared HIV-1 Tat protein, Partidos et al. (62) were able to induce robust anti-Tat antibody responses in BALB/c mice following transcutaneous delivery in combination with CT. Moreover, the anti-Tat antibodies were capable of neutralizing Tat activity in a chloramphenicol acetyltransferase assay for transactivation. The TCI with combined Tat  CT also elicited IgA antibodies that were detected in vaginal washes, a result consistent with previous observations that TCI can elicit mucosal immunity to coadministered antigens (13). It should be noted that when Tat protein was applied topically without an adjuvant, no anti-Tat antibody responses were detected, highlighting the importance and use of CT as an adjuvant on Tat immunogenicity. Given the potential of developing T-cell immunity to HIV, iTCI was found to induce strong Tat-specific T-cell responses as measured by secretion of high levels of IL-2, IFN-g, and IL-6.

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Measles Virus A recent phase I trial involving a live-attenuated measles vaccine, ROUVAX1, was performed to compare subcutaneous versus TCI routes of vaccination. Etchart et al. (63) demonstrated that TCI using a skin patch is acceptable and safe, and that the level of pain and discomfort was lower for the transcutaneous versus subcutaneous procedures. In regard to immunogenicity, they observed that serum MV-specific neutralizing antibodies were induced by the subcutaneous route but not by TCI. On the other hand, the TCI route increased the levels of MV-specific salivary IgA, whereas no increase was obtained after subcutaneous immunization. Thus, TCI with the live virus vaccine induced mucosal immunity, supporting the concept that homing of antigen-specific IgA B cells to the mucosal site (i.e., salivary glands) distinct from initial site of immunization had occurred. In addition to the increase in MV-specific salivary IgA levels, there was also a rise in the frequency of MVspecific IFN-g producing cells after transcutaneous vaccination. For example, 9 out of 12 of TCI recipients versus 3 out of 9 individuals of the subcutaneous group exhibited a detectable increase of MV-specific IFN-g production. These results suggest that TCI is more efficient at inducing a virus-specific T-cell response, and offers the advantage of inducing specific mucosal IgA antibodies; this may be of interest for protection against respiratory virus infections. At the moment, the reasons for the discrepancy in the humoral responses between the transcutaneous and subcutaneous groups are not clear; however, it should be pointed out that no coadministered adjuvant, such as LT or CT, was used in the TCI protocol. Human Papillomavirus In addition to TCI applications involving large biomolecules (protein, whole viruses, etc.), smaller size products, such as peptides, have also been used to elicit antigen-specific CTL and Th responses. Persistent infection with human papilloma virus is responsible for more than 50% of cervical cancers worldwide (64). The strategy for HPV vaccines is to induce cell-mediated immunity against the HPV-16 E6 and/or E7 oncoproteins, which are constitutively expressed within the tumor cells. Recently, Dell et al. (65) have immunized mice transcutaneously in the presence of CT and CpG with an HPV-16 E7-specific peptide consisting of a combination of a CTL, Th, and B-cell epitope. The multi-epitope HPV peptide induces strong functional E7-specific CTL responses. Moreover, the TCI peptide immunization protected mice against tumor growth following challenge with HPV-16 E7-positive tumor cells. These data suggest the possibility that TCI might be explored as an alternative to existing HPV vaccination strategies. Virus-Like Particle (Rabbit Hemorrhagic Disease Virus) Virus-like particles (VLPs) are actively being pursued as vaccine candidates for influenza, human papilloma virus, and hepatitis. Recently, VLPs have been licensed for human use against cervical cancers (64). TCI application of a VLP has been reported by Young et al. (66). They used a VLP derived from rabbit hemorrhagic disease virus and tested whether it can induce mucosal IgA as well as a Th1 response in the presence of coadministered CT and CpG motifs. An effective vaccine against mucosal infections, particularly to viruses, should induce both mucosal IgA and Type 1 T cells. After several TCI boosts, they were able to detect increased productions of mucosal IgA in vaginal lavages of mice. Moreover, the VLP delivered by TCI induced a high level of IFN-g, which is

usually associated with a Type 1 immune response. Given the initial success of their TCI study, they are exploring the transcutaneous route of delivery by VLP incorporating welldefined bacterial epitopes. Pandemic Influenza In the event of an influenza pandemic, a more immunogenic or dose-sparing vaccine, as well as an effective vaccine delivery technology, is desirable. Current vaccine candidates, such as the recombinant H5N1 hemagglutinin (rHA) protein or whole, inactivated H5N1-based reassortant virus strains have been poor immunogens. Garg et al. (67) have explored a novel transdermal patch technology to deliver baculovirus-expressed recombinant H5 HA through mouse skin via an electroporation device. The rH5 HA protein was transcutaneously delivered with one of two adjuvants: CpG (ODN), which is a TLR9 ligand, and resiquimold, a TLR7 ligand. The use of CpG (ODN), but not resiquimold, significantly increased the HI titers to levels comparable to control mice immunized by intraperitoneal injection. Moreover, the TCI delivery induced protective humoral responses against lethal challenge with a highly pathogenic avian H5N1 virus strain. Their findings suggest that the immunogenicity of current pandemic vaccine candidates may be improved by the use of adjuvant and by needle-free TCI. We have previously shown that an adjuvant patch containing LT placed over a vaccine injection site can greatly augment the immune response to the injected antigen whether injected intramuscularly, subcutaneously, or intradermally. In preclinical studies involving mice and guinea pigs, the LT patch has been demonstrated to provide 10- to 100-fold dose sparing for an rHA protein representing the A/Vietnam/1203/ 2004 strain of H5N1 influenza virus (27). A dry LT patch is currently under clinical investigation with an inactivated H5N1 split-virion vaccine candidate. Interim results of a phase I/II clinical trial using LT as an IS patch to enhance the immune response to the injected H5N1 vaccine is described in section ‘‘LT Dry Patch: Improved Immune Responses to Injected H5N1 Vaccine.’’

TCI Applications for Miscellaneous Diseases Melanoma Yagi et al. (68) have applied antigenic melanoma-associated peptides onto the skin of human patients with melanoma. Following SC disruption by glue treatment, the percutaneous peptide immunization (PPI) activated the LCs in the epidermis, and caused their maturation and emigration to the lymphoid organs. The LCs bearing peptides induced generation of IFN-g and potent circulating CTLs. The PPI was beneficial and effective in patients with advanced melanoma, as evidenced by the reduction in the lesion size and suppression of further tumor development in four of seven patients. Interestingly, no adjuvant was used in the PPI study. Apparently, the skin barrier disruption with the strong glue not only enhanced the permeability of the melanoma-associated peptides but also activated and fully matured the epidermal LCs for antigen presentation. Alzheimer’s A recent vaccine development for Alzheimer’s disease (AD) is in the form of a skin patch. Previously, an injectable b-amyloid (Ab) peptide vaccine proved effective in clearing brain plaques in an AD mouse model; however, when used in a phase I trial,

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it ended in tragedy when a small percentage of human subjects developed brain inflammation and died. Nikolic et al. (69) have now developed a transcutaneous Ab vaccination approach. By using a TCI protocol with an Ab1–42 peptide  CT on mice skins, they observed high-titer anti-Ab antibodies (mainly of the IgG1 class, indicative of Th2 response) and anti-Ab-specific splenocyte immune responses in transgenic mice specially bred to develop AD. Their TCI vaccination resulted in significant reduction of cerebral amyloidosis and was not associated with deleterious side effects, including brain T-cell infiltration or cerebral microhemorrhage. The conclusion was that the vaccine skin patch induced a Th2 type of immune reaction, which was predicted to have less likelihood of producing the autoimmune reactions, such as seen in the phase I trial. It is likely that the parenteral Alzheimer’s vaccine that gave rise to the autoimmune reactions seen in a phase I trial elicited Th1 immune responses.

CLINICAL PRODUCT DEVELOPMENT: DRY PATCH FORMULATION, MANUFACTURING, AND THERMOSTABILITY The recent successes of TCI prompted the development of a dry, excipient-stabilized vaccine/adjuvant patch delivery system that is efficient and user friendly. Ideally, the vaccine patch formulation should also possess an adequate thermostability profile to allow storage and use at room temperature, and to withstand temperature fluctuations during shipment and distribution for clinical use. Toward this end, Intercell scientists have made significant advances in developing a late-stage product using LT as a candidate for an ETEC traveler’s vaccine, as well as its use as an immunostimulatory patch for dose sparing in pandemic and seasonal influenza. As discussed further, the technical progress made in developing a commercial patch application for LT has been extended to develop a dry patch formulation for seasonal influenza.

Dry Patch Manufacturing Overview Once the optimal formulation has been established, the dry patch is prepared. This is accomplished by dosing a small volume of the drug substance blend formulation onto an absorbent patch matrix (typical surface area of 1 and 3 cm2) followed by moderate drying in an air convection oven. The antigen-dosed patch matrix is then assembled with an occlusive backing, release liner and adhesive overlay as depicted in Figure 1B. The occlusive backing provides an impermeable barrier between the active drug formulation and the adhesive overlay, and, more importantly, traps transepidermal water loss (TEWL) from the pretreated skin area to hydrate the dry patch. The release liner covers and protects the active patch formulation during storage, and is sealed to the adhesive overlay to prevent moisture and air ingress into the patch assembly. Once the release liner is removed, the adhesive overlay adheres the patch to the skin.

Patch Dryness and Reconstitution on Skin To maintain the state of dryness, the patch assembly is placed in a foil pouch, heat-sealed under nitrogen atmosphere, and stored at 28C to 88C. The patch dryness is assessed by moisture content or by its water activity. The latter parameter is commonly used by microbiologists and food technologists to assess the safety and quality of food products (70–72). According to USP

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<1112>24, when the water activity, aw, of a product decreases below 0.60, microorganisms cannot obtain the water needed to support their growth. Most bacterial growth is inhibited below aw & 0.91; most yeasts cease growing below aw ¼ 0.87, and most molds do not grow below aw ¼ 0.80. The aw of Intercell’s dry patch is around 0.35  0.03, significantly lower than the absolute limit of microbial growth of aw ¼ 0.60. Thus, the dry patch formulation will not support microbial growth during storage. Unlike a lyophilized vaccine vial formulation, which is reconstituted by a manual multistep process (i.e., syringe injection of diluent into vial, subsequent mixing of components in vial, measured vaccine volume withdrawal in syringe, human injection), the dry patch formulation is simply hydrated by the TEWL entrapped under the patch occlusive backing after patch application on pre-treated skin. Because of its inherent hydroscopic property, the dry patch formulation is readily hydrated by TEWL, and the vaccine antigen and/or adjuvant, in turn, are efficiently solubilized and released from the patch onto the skin. Patch hydration enhances passive antigen delivery since the early stages of the solubilization process result in saturated concentrations of antigen and/or adjuvant on the skin surface. This higher concentration gradient provides the thermodynamic driving force to deliver more antigen and/or adjuvant into the skin. The effectiveness of skin delivery by a dry versus wet patch vaccine is further described in section ‘‘LT Dry Patch: Improved Immune Responses to Injected H5N1 Vaccine’’ later in the text.

Patch Thermostability: LT and Flu Dry Patches as Examples Intercell’s proprietary patch formulations impart excellent stability for dry patches stored at refrigerated and ambient conditions. As shown in Figure 2, the dry LT patch met the Y-1 potency activity specification for all test points (i.e., 22 months at 2–88C and for 6 months at 258C) and the label claim specification for LT content, as measured by SE-HPLC [i.e., 22 months at 2–88C and 9 months (endpoint of accelerated testing) at 258C]. At 408C, the LT content met specification after one month of storage; the losses of LT were significant thereafter. The stability of the dry TIV patch lot at 58C, 258C, and 408C is shown in Figure 3, with respect to HA content as measured by the SRID assay for the A/New Caledonia/20/ 99 (H1N1), A/Wyoming/3/2003 (H3N2), and B/Jiangsu/10/ 200 strains. At 58C and 258C, the HA content trended above the specification requirement, thus indicating that the dry TIV patch is stable for 12 months. At the elevated temperature of 408C, the HA potency for all three influenza strains met specification after one-month storage; however, the loss of HA was significant thereafter, especially for B/Jiangsu and A/New Caledonia virus strains. The dry LT and TIV patches were also exposed to thermal cycling conditions to assess their tolerance to temperature excursions that may be encountered during shipping and distribution. After 12 days of thermal cycling between 208C and 258C (intended for product to be stored refrigerated), and between 208C and 408C (intended for product to be stored at room temperature), there was no statistically significant loss in product quality observed for both the LT and TIV patches (data not shown). Since the packaged products could tolerate subfreezing and elevated temperatures, the thermal cycling studies suggest the possible use of expanded shipping conditions for the LT and TIV patches (beyond 2–88C).

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Figure 2 Stability profile of LT dry patch (50 mg dose) at 58C, 258C, and 408C as assessed by LT content (by SE-HPLC, top panel) and potency (by Y-1 cell assay, bottom panel). Abbreviation: LT, labile enterotoxin.

pig immunogenicity study confirms that complex macromolecules, such as split-inactivated flu virus antigens, can be delivered effectively across pretreated skin, and that serum anti-flu IgG antibody levels can be augmented by coadministration with LT adjuvant.

Whole Influenza Virus Patch

Figure 3 Stability profile of HA content for dry trivalent inactivated influenza vaccine patches (45 mg HA total dose) stored at 58C, 258C, and 408C. The HA content was extracted from dry patch and assessed by single radial immunodiffusion assay for each viral strain. Abbreviation: HA, hemagglutinin.

INTERCELL DRY PATCH VACCINES: TESTING IN ANIMAL MODELS Trivalent Inactivated Split-Influenza Vaccine (TIV) Patch Using patch-stabilizing platform technology, Intercell prepared a dry patch for seasonal influenza and used it along with the SPS device (for skin pretreatment) in guinea pigs, whose SC and epidermis are similar to human skin. As shown in Figure 4, primed animals immunized with a TIV patch containing 5 mg LT and 5 mg HA each of the three split virus influenza antigens (A/Wyoming/3/2003, A/New Caledonia/20/99, B/Jiangsu/ 10/2003; 15 mg HA total) achieved similar or higher levels of anti-influenza serum IgG antibody titers than animals injected intramuscularly with the same HA antigen dose. The guinea

We have evaluated the immunogenicity of a whole H3N2 virus applied on a patch at 0.01, 0.10, and 1.0 HA dosages. As shown in Figure 5, the 1.0 mg HA whole virus patch (63,529 ELISA units) proved highly immunogenic, giving a level of anti-H3N2 ELISA antibodies comparable to that of a control group (61,904 ELISA units) immunized IM with a split-influenza monovalent vaccine containing 15 mg of H3N2 HA. Apparently, the whole virus behaves as a strong immunogen on the skin; this could be due to its multimeric and/or particulate nature that renders efficient uptake and antigen processing by the skin LCs. When the whole virus patch was given with LT, the anti-H3N2 titer was greatly enhanced. Nearly 150-fold dose sparing was achieved when the 0.10 mg HA whole virus patch was given with 5 mg of LT (82,495 ELISA units) in comparison to the IM control group receiving 15 mg H3N2 HA dose (61,904 ELISA units). These data show that it is feasible to obtain an improved overall immune response by using an adjuvant and an ideal influenza antigen such as a whole-virus particle.

LT Immunostimulatory Patch for Pandemic and Seasonal Flu An early clinical study showed that LT could be used as an IS patch, along with a conventional injected vaccine, against influenza disease (24). This study used a liquid formulation of LT that was applied onto a gauze pad placed over the pretreated skin injection site. The ‘‘wet’’ LT patch, used at the time of injection, resulted in an augmentation of the antiinfluenza immune response in the elderly (human subjects

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Figure 4 TCI delivery of TIV antigens via dry formulated patches compared to intramuscular injection, as measured by hemagglutinationinhibition titers in guinea pigs. For TCI, the guinea pigs (N ¼ 6 per group) were immunized with dry TIV patch containing 15 mg total hemagglutinin with and without 5 mg of labile enterotoxin. The geometric mean for each viral strain (A/Wyoming/3/2003, A/New Caledonia/20/ 99, and B/Jiangsu/10/2003) is represented by the dashed horizontal bar. Abbreviations: TCI, transcutaneous immunization, TIV, trivalent inactivated influenza vaccine.

Figure 5 Transcutaneous immunization patch delivery of whole H3N2 influenza virus in guinea pigs as measured by serum anti-H3N2 IgG titers. The guinea pigs (N ¼ 6 per group) were transcutaneously immunized with patches containing 0.01, 0.1, and 1.0 mg HA (as determined by SRID assay of the whole virus) with and without 5 mg of labile enterotoxin. The control group (N ¼ 6) was immunized intramuscular with 15 mg HA of a monovalent split-virus H3N2 vaccine. Abbreviation: HA, hemagglutinin.

over 60 years) receiving their recommended annual influenza vaccination (24). As a prelude to human use, the dry LT patch was evaluated in preclinical studies as a leading candidate for a dose sparing/enhanced immunity strategy for pandemic and seasonal flu vaccines. As shown in a mouse model (Fig. 6), the LT-IS patch provided up to 100-fold dose sparing for a

pandemic flu vaccine candidate (27). In this study, mice were immunized ID with an rHA protein representing the A/Vietnam/ 1203/2004 (H5N1) strain at 1.5, 0.15, and 0.015 mg. Half of each group received a 10 mg LT-IS patch placed over the injection site, while the other half received a placebo patch. After a two-dose regimen, the rHA immune responses in the group receiving the 0.015 mg dose with the LT-IS patch had an

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Figure 6 Dose sparing in mice immunized with rHA protein representing A/Vietnam/1203/2004 (H5N1) (potentially pandemic) strain. The LT-IS patch provided up to 100-fold dose sparing for the pandemic flu vaccine candidate. Mice received two intradermal immunizations 14 days apart of rHA and LT-IS patches. ELISA serum anti-rHA IgG titers are shown. Groups receiving LT-IS patches had statistically greater titers than groups receiving placebo patches. Abbreviations: LT-IS, labile enterotoxin–immunostimulatory; rHA, recombinant hemagglutinin

antibody response (47,491 ELISA units) comparable to the group receiving the 1.5 mg dose alone (20,142 ELISA units); thus suggesting that a 100-fold reduction in vaccine may be achievable when the vaccine is delivered intradermally with

the LT-IS patch. Data collected in the same study suggest that a 10-fold reduction in vaccine can be achieved for mice immunized by IM injection (data not shown). In addition to mice, a similar dose-sparing range of the H5N1 rHA protein was obtained in a guinea pig study. Taken together, the data in the two animal models show that the LT-IS patch can achieve 10- to 100-fold dose sparing. In another study, the LT-IS patch effect was evaluated in mice using four different manufacturers’ trivalent influenza vaccine products (Fig. 7). The mice were immunized twice, two weeks apart, by ID injections of 4.5 mg of HA dose per virus strain with an LT-IS or placebo (no LT) patch placed over the injected site. The anti-A/New Caledonia serum IgG titers are shown as representative of IgG responses in all three virus strains. The mice receiving the LT-IS patches had significantly higher anti-influenza IgG titers than those receiving the placebo patches. Moreover, the influenza vaccines were enhanced equivalently among all four manufacturers’ products, thus demonstrating that the adjuvant effect is not antigendependent, and reinforcing the idea that an LT-IS patch can be considered as a universal dose sparing or immune enhancement strategy for pandemic influenza vaccines.

CLINICAL STUDIES WITH INTERCELL DRY LT PATCH IN HUMANS Dry Versus Wet Patch for LT Due to its inherent hygroscopic properties, the dry LT patch formulation is readily hydrated by the TEWL resulting from skin pretreatment with the SPS device. Solubilization of antigen and adjuvant readily occurs, resulting in high concentrations of the antigen and adjuvant at the surface of the skin. The high

Figure 7 The LT-IS patch combined with different injectable seasonal influenza vaccines produced statistically greater serum ELISA IgG titers in mice than those receiving a placebo patch. Mice received two intradermal immunizations, 14 days apart of the commercially available seasonal flu vaccines and the LT-IS patches. Only anti-A/New Caledonia serum IgG titers are shown. Abbreviation: LT-IS, labile enterotoxinimmunostimulatory.

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Figure 8 Anti-LT IgG titers in human volunteers receiving either a ‘‘wet’’ or ‘‘dry’’ patch containing 50 mg LT on Day 0 and Day 21. The LT IgG titers were measure on Days 7, 14,21,28,35, and 42 and compared to baseline titers. The mean fold rise in LT IgG is indicated by the triangles for the dry patch group and by the hexagonals for the wet patch group. Abbreviations: LT, labile enterotoxin.

concentration gradient provides the thermodynamic driving force to deliver the antigen and adjuvant into the skin. The hypothesis that a dry patch could deliver antigen as efficiently as a wet patch was tested in the clinic. In this study, half of the subjects received a wet patch in which an LT solution was pipetted onto a gauze patch placed over the SPS-treated skin, while the other half received the dry formulated LT patch. Altogether, 160 human volunteers were given a 50 mg LT dose in either a wet or dry patch. Each subject was immunized twice on days 0 and 21, and blood samples were collected on days 7, 14, 21, 28, 35, and 42 for determination of LT IgG titers. As indicated in Figure 8, factorial analysis using LT IgG titers and fold rise (ratio of postimmunization to baseline titers) showed that the dry patch produced significantly higher titers and fold ratios of LT IgG than the wet patch at all time points from day 14 onward. The clinical data are consistent with preclinical studies showing that the dry LT patch is more efficient than wet patch for vaccine delivery.

LT Dry Patch–Prevention and Reduction of Travelers’ Diarrhea Illness LT is a key virulence factor in most (ETEC) strains, and ETEC is the most common pathogen associated with travelers’ diarrhea (5–10 million LT-related ETEC cases, annually) and is a major cause of weanling infants’ diarrhea (200 million annual cases). There are substantial data indicating that prior ETEC clinical infections lead to resistance to subsequent clinically apparent infections; rises in anti-LT are also evident following many clinical ETEC infections (73–75). LT can elicit robust systemic and mucosal immune responses when delivered transcutaneously and LT delivery to the skin provides a unique safety margin, since the toxin does not penetrate into the vascularized

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dermis but is efficiently captured and carried out of the skin by dendritic cells to the key components of the immune system. A recent report documents the safety and robust immune response generated in a phase I, double blind, live organism challenge study using LT as an immunogen delivered in a wet patch format (76). In the study, adult volunteers received three applications of a 50 mg LT patch or a placebo patch. Two weeks following the third dose, both the vaccinee and placebo groups were orally challenged with 6108 LTþ/STþ ETEC organisms (76). The clinical results indicated that the wet LT patch produced strong systemic and mucosal anti-LT responses. Even after one dose, 97% of the vaccinees seroconverted (>fourfold rise in anti-LT IgG or IgA titers), and the levels of anti-LT IgG and IgA were higher than those of the placebo group following oral challenge with the virulent ETEC strain. In addition, fecal anti-LT IgG and IgA, as well as LT-specific antibody IgG and IgA secreting cells, were detected in the vaccinees after one dose. The LT-specific ASCs were boosted by the challenge, suggesting that the mucosal immunity induced by TCI could be boosted by gut challenge. Overall, TCI did not prevent moderate to severe ETEC disease in the challenge model. However, TCI did mitigate the severity of diarrhea in subjects who became ill. Vaccinees had significantly lower rates of stooling, reduced stool weights, longer time to onset of disease, and decreased need for IV therapy, suggesting that the anti-LT immunity neutralized the effects of the toxin at the level of the gut. One interpretation of the results is that oral challenge with the moderate dose of virulent ETEC overwhelmed the immune response elicited by the LT patch vaccine. A high dose is often given in experimental volunteer challenge studies to assure a high attack rate in the unvaccinated control subjects. Studies on contaminated foods suggest that in nature the usual infective inoculum ingested may be 2 to 3 logs lower dose than the dose of ETEC administered in the challenge study. Despite the severe oral challenge, the phase I study suggests that LT delivered on the skin can ameliorate the severity of ETEC illness and that the vaccine effect may be amplified in the field, where the ingested ETEC dose would most likely be lower. Subsequent to the phase I challenge trial, the dry LT patch, along with the SPS device, was evaluated in a randomized, double blind, placebo-controlled field trial (15,77). In this study, 170 United States travelers to Guatemala and Mexico were vaccinated twice, two weeks apart, with either a 37.5 mg LT dry patch or a dry placebo patch in a 1:2 ratio. Significantly higher anti-LT IgG and IgA titers were observed in the vaccinee versus placebo groups at the time of arrival in country (IgG 8,914 vs. 526 EU; IgA 398 vs. 49 EU) and at exit (IgG 11,060 vs. 649 EU; IgA 442 vs. 64 EU) with seroconversion rates of 92% and 78% for anti-LT IgG and IgA, respectively. The dry LT patch conferred 76% vaccine efficacy against moderate and severe diarrhea and 84% efficacy against severe diarrhea from any cause. Moderate diarrhea was graded as four to five loose stools in a 24-hour period, whereas severe diarrhea was defined as more than six loose stools per 24-hour period. As shown in Figure 9, the number of cases of moderate or severe diarrheal disease of any cause was significantly greater in placebo recipients than in vaccinees. Moreover, in those who developed diarrhea, the duration of illness was significantly reduced for the vaccinees (0.45 day) versus the placebo groups (2.1 days) as well as the stool frequency: 3.7 for vaccinees versus 10.5 for placebo group. Although not statistically significant, the frequency of new-onset irritable bowel syndrome, a long-term

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Figure 9 Severity of diarrheal episodes and number of stools for all subjects who had diarrhea. Cumulative stools from individual episodes for the vaccinee and placebo groups are shown. Moderate diarrhea was considered as four to five loose stools in a 24-hour period, whereas severe diarrhea was graded as more than six loose stools per 24-hour period. In this study, 170 U.S. travelers to Guatemala and Mexico were vaccinated twice, two weeks apart, with either a 37.5 mg labile enterotoxin dry patch or a dry placebo patch in a 1:2 ratio.

consequence of travelers’ diarrhea, was about three times greater in placebo than vaccine recipients. On the basis of the expected 10% to 20% incidence of LT-secreting ETEC in these countries, the protective efficacy of 76% against moderate and severe diarrhea against any cause was unexpected and greater than predicted, suggesting that the immune response to LT in a dry patch somehow extends protection beyond LT-secreting ETEC. In this regard, it is noteworthy that in three field studies, oral CTB, which elicits antitoxin that cross neutralizes LT, has provided cross protection against Salmonella and Salmonella/LT ETEC mixed infections (78), against Campylobacter (79), and against LT- and heat stabile toxin (ST)-containing ETEC (80). Together, the data suggest that an LT patch may be able to confer protection against other pathogens that cause travelers’ diarrhea, in addition to LT-producing ETEC. It has been hypothesized that immunity to LT blocks the conditioning of the gut wall for enhanced enteric pathogenicity caused by other intestinal disease-causing microorganisms; this hypothesis warrants further study (81).

LT Dry Patch: Improved Immune Responses to Injected H5N1 Vaccine The dry LT patch was recently evaluated as a strategy to improve the immune response rates in subjects who received an injected split-virion pandemic influenza vaccine candidate (reassortant A/Vietnam/1194/04 NIBERG strain). In this Intercell study, 500 healthy adult subjects received either a placebo injection or one of three antigen doses (5, 15, and 45 mg H5N1 HA) with and without the LT-IS adjuvant patch. Interim results of the 500-subject clinical trial indicate that for all vaccine dosage levels, LT-adjuvanted groups manifested higher anti-H5N1 HAI GMTs, higher fold rises, and higher rates of seroconversion and seroprotection than the corresponding nonadjuvanted groups. These data clearly

demonstrate the adjuvant effect of the LT patch (unpublished data). More importantly, the trial found that a single 45 mg vaccine dose coupled with an LT patch adequately met all three CHMP criteria (82) for a licensed pandemic vaccine—that is, more than 2.5-fold rise in geometric HAI titer, more than 40% seroconversion, and more than 70% seroprotection. With respect to seroprotection, 73% of these patients in the 45 mg vaccine  LT patch group recorded a postvaccination HAI titer 1:40, as compared to 49% of those who received the vaccine alone (p < 0.0001). This significant difference exceeds guidance provided by the FDA for demonstrating evidence of an adjuvant effect. Research to date has indicated that H5N1 influenza vaccine candidates are inherently poor immunogens. Larger doses (e.g., 90 mg) than the 15 mg present in seasonal influenza vaccines, as well as two-dose vaccine regiments (with less splitvirion H5N1 antigen) requiring special or proprietary oil-inwater emulsion-based adjuvants, have been cited in the literature as achieving protective levels against bird influenza pandemic viruses (83–85). The best protection level reported for a single dose was 58%, for subjects vaccinated with a 30 mg H5N1 dose given in combination with a proprietary oil-in-waterbased adjuvant (83). This protection rate is lower than the 73% level achieved with the LT-IS patch. So far, the only approved vaccine in the United States for the H5N1 influenza virus requires two 90 mg doses to generate a protective level in about 45% of vaccinated individuals. Intercell’s phase I/II interim results demonstrate that a single vaccine dose involving the placement of an LT patch over the injection site of a 45 mg HA dose (inactivated split A/Vietnam/1194/04 NIBERG strain) can generate an immune response that exceeds the protective levels of the licensed vaccine, thus potentially eliminating the need for a second round of vaccination and reducing the amount of H5N1 vaccine by fourfold.

SUMMARY AND FUTURE DIRECTION Needle-free delivery of vaccines remains a high priority for the World Heath Organization. TCI is an alternative route of vaccine delivery that bypasses the need for needles and syringes. Basic principles of TCI have been demonstrated and cover a wide range of bacterial and viral products in both animal and human studies. Findings from Intercell studies, as well as from other groups of investigators indicate that: l

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TCI delivery of proteins and vaccine antigens to the skin epidermis can induce systemic, mucosal, and cell-mediated immune responses. Antigen-specific CTL immune responses can also be induced by TCI vaccinations with viral and tumor peptide antigens. Pretreatment of SC improves efficiency for skin delivery of vaccine antigens and adjuvants. Delivery of large entities, including whole organisms, to the epidermis is possible once the SC is disrupted. LT and other adjuvants delivered to the skin markedly enhance immune responses to vaccine antigens. The adjuvant can be coadministered with vaccine antigen in a TCI patch or delivered separately from antigen as an IS patch. Fewer doses and dose sparing are possible. Biological products, such as LT and trivalent influenza vaccines, have been formulated and stabilized in dry patch format. Intercell’s proprietary patch formulation is readily hydrated by TEWL to allow efficient delivery of antigens onto the skin by passive diffusion.

Chapter 40: Advances in Transcutaneous Vaccine Delivery l

l

l

l

The dry patches exhibited long-term stability at 58C, more than six-month stability at 258C and proved thermostable during temperature excursions. Elimination of cold-chain storage is possible for patch distribution. In a phase II field study, Intercell’s dry LT patch was safe and immunogenic, and protected travelers against moderate and severe diarrhea. Vaccine efficacy against moderate and severe diarrhea from any cause was 76% (p ¼ 0.007) and against severe diarrhea was 84% (p ¼ 0.03). Interim results of a clinical trial suggest that Intercell’s dry LT patch can improve the immune response rates in humans receiving an injected split-virion pandemic influenza vaccine candidate. A single 45 mg H5N1 vaccine dose coupled with an LT patch adequately met all three CHMP criteria for a licensed pandemic vaccine (unpublished data). The Intercell SPS device and the dry patch formulation can be combined in a TCI delivery package system that is marketable and less expensive to manufacture. Moreover, it can be put in an envelope and mailed for mass distribution and self-administration.

Although significant applications and advances have been made in TCI technology within the last several years, there are still opportunities for further research and development. One particular area for active research and development involves optimal and stable patch formulations compatible to the physicochemical properties of large molecular weight entities, for example, formalin-treated cross-linked proteins, live-attenuated or killed viruses, VLPs, killed bacteria, etc. These large macromolecules require formulations to enhance their solubilization (release) from the dry patch to allow their efficient penetration into the skin epidermis to induce systemic and mucosal immune responses.

ACKNOWLEDGMENTS The authors gratefully acknowledge Dr Barbara Butler and Wanda Hardy for their assistance with review and preparation of the manuscript.

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41 Rationalizing Childhood Immunization Programs: The Variation in Schedules and Use of Combination Vaccines Andrew J. Pollard Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Children’s Hospital, Oxford, U.K.

Kathryn M. Edwards Department of Pediatrics, Division of Pediatric Infectious Diseases, Pediatric Clinical Research Office, Vanderbilt University School of Medicine, Nashville, Tennessee, U.S.A.

Bernard Fritzell Wyeth Vaccines Research, Scientific and Clinical Affairs, Paris, France

HETEROGENEITY OF IMMUNIZATION SCHEDULES Immunization is one of the most important achievements in public health. Vaccines have substantially contributed to reductions in global morbidity and mortality due to infectious disease over the past decades. Recommendations on vaccination are guided by the risk of disease (incidence and severity), the expected protection conferred by the vaccine (benefit), and the potential adverse events of the vaccine (risk). For any given vaccine, the immunization schedule used and the age groups targeted may vary substantially from one region of the world to another or, as is particularly the case in the European Union (EU), even among countries of the same region. Many factors contribute to the diversity in immunization policies across the world, including the nature of the immune response to a vaccine, the disease burden and the fraction of that burden that is vaccine preventable, the cost of vaccination (price of the vaccine and delivery of vaccination), the capacity and competence of health care provision, and other societal factors (perception of the disease and of the benefits and risks of vaccination). The immunization schedule recommended by the WHO Expanded Program on Immunization (EPI) is uniformly adopted by nearly all developing countries. In contrast, there is great diversity in immunization schedules used in industrialized countries and within particular regions of the developed world, despite the fact that the medical and epidemiological differences of vaccinetargeted diseases seem limited. For effective protection, immunization should begin at an age, which allows protective immune responses to be initiated safely and before the peak age-specific incidence of the disease. The duration of protection is also a criterion that influences the immunization regimen, of particular relevance when considering a booster dose or doses. In addition, for diseases with person-to-person transmission, vaccines may not only protect vaccinees from becoming infected or ill, they may also reduce the transmission of the targeted pathogen in the community and offer indirect (‘‘herd’’) protection to those who have not been immunized. The magnitude of this indirect effect may be an

important component of the overall effectiveness of an immunization program so it is also important to consider that herd immunity might be influenced by the vaccination schedule. Regulatory agencies, such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMEA), or the national regulatory agency (NRA) for countries in other regions of the world, grant a marketing authorization for a new vaccine that considers the quality of the product and the adequacy of safety and efficacy data leading to a favorable risk/benefit ratio. Pre-licensure clinical documentation allows definition of the target age group and establishes the number of doses for the primary series, the interval between doses, and the need for a booster dose to achieve effective clinical protection. It also provides information as to whether simultaneous administration with the other vaccines recommended for the same age group would also be safe and effective. Once a vaccine is licensed, individual governments establish national policy on childhood immunization. For example, recommendations on immunization in the United States are issued by the Advisory Committee on Immunization Practices (ACIP), an advisory group to the U.S. Centers for Diseases Control and Prevention (CDC). In the case of private sector practitioners in the United States, the committee on infectious diseases of the American Academy of Pediatrics publishes recommendations on vaccination (1), and guidance is provided by the American Academy of Family Practice (AAFP) for pediatric vaccines, while the AAFP and the American College of Physicians supply recommendations for adult vaccination schedules. Most pediatric schedules are designed to coincide with DTP infant immunization visits. For example, current recommendations for DTP vaccine in the United States include three doses given at two, four, and six months of age, followed by a booster at 12 to 15 months. Subsequently introduced vaccines such as hepatitis B (HB) (for the doses administered after birth), Haemophilus influenzae type b conjugate (Hib), inactivated poliovirus vaccine (IPV), pneumococcal conjugate vaccine, and rotavirus have been introduced to coincide with the DTP schedule, although the number of doses administered

Chapter 41: Rationalizing Childhood Immunization Programs

may vary according to the vaccine. In addition, one dose of measles, mumps, rubella (MMR) and varicella (V), either combined or separately administered, is given at 12 to 15 months followed by a second dose at 4 to 6 years. Two doses of hepatitis A (HA) vaccine, 6 months apart, are recommended for all children between 12 and 23 months. In the EU, the heterogeneity of vaccination schedules has arisen for essentially historical reasons. In the past, vaccines were registered in an individual country according to licensing requirements that might vary substantially from country to country. Although licensure in a given country is still possible under restricted conditions, the marketing authorization of a new vaccine in the EU currently follows a unified process, either through mutual recognition or a centralized procedure. Nonetheless, there is no European legislation that reinforces harmonization of immunizations programs across the 27 countries of the EU. Public health policy, including immunization policy, is the responsibility of the member state under the principle of subsidiarity. Consequently, each country relies on a national advisory body that makes recommendations on immunization policies to the Ministry of Health [e.g., the Joint Committee on Vaccination and Immunisation (JCVI) in the United Kingdom, or the Sta¨ndige Impfkommision am Robert Koch Institut (STIKO) in Germany]. In addition, within a country, the level of centralization in public health policy and funding influences the recommendations and implementation of immunization programs. For example, in Spain, each ‘‘autonomous region’’ decides on its own immunization program, and recent recommendations on group C meningococcal (MenC) vaccines illustrate the lack of harmonization in this country as a result of regional decision making. Furthermore, in countries within the WHO European region, immunization policy is influenced by policies and objectives that have been set by WHO Regional Office for Europe (2). Within the EU, substantial differences in the perception of the disease burden by the recommending authorities may exist because there is no formal standardized framework for assessing the burden of disease across the EU. Nonetheless, possible epidemiological differences in the features of a vaccinepreventable disease are unlikely to explain the existing variations in schedules of vaccination. In addition, comparison of the effectiveness of different immunization programs across countries in the region may not be possible due to varying surveillance of vaccine-targeted diseases, making it difficult to justify a change in immunization practices to harmonize recommendations. The benefit of simultaneous administration with an already established immunization regimen has usually driven the decision about introduction of a new vaccine in the established schedule and has perpetuated the diversity in immunization schedules set for the vaccines that were first recommended. The existence of entrenched schedules in different jurisdictions also guided introduction of combination vaccines for use in these established schedules. The attitudes of health care providers and of the public to established vaccinations may also contribute to the absence of harmonization. Finally, perception of the risk of vaccine-targeted disease by the public can vary. In public health and primary care facilities, existing wellbaby visits are used to deliver immunizations, but these schedules are heterogeneous across the EU. Recommendations for booster immunization at school age may be facilitated when school-based programs are mandated to deliver immunization at school.

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As mentioned above, notable diversity exits in pediatric immunization practices in the EU, which is described in Table 1 for the DTP vaccine (3). A primary infant series consisting of either two or three doses is generally completed by six months of age. Among countries that have adopted a three-dose primary immunization schedule, the interval between dosing may be either one month (accelerated schedule such as the 2, 3, and 4 months regimen in place in France, Germany, and the United Kingdom) or two months (2, 4, and 6 months, as in Portugal and Spain). Timing for booster vaccinations is also variable; any time in the second year of life for most countries and at school entry in others. When a two-dose primary series is recommended, as in the Nordic region and Italy, DTP is usually given at three and five months with a third dose (booster) at 12 months. In nearly all Latin American countries, the primary series for DTP given either as a stand alone or as a combination (DTPHepB and Hib or DTP-Hib) consists of three doses given at two, four, and six months followed by booster dose at 12 to 18 months (4). Three doses of oral poliomyelitis vaccine are scheduled at two, four, and six months of age, with a fourth dose, in most countries of the region, between 12 and 18 months. With the exception of Haiti where one dose of measles vaccine is given at nine months, one dose of MMR vaccine is generally administered at one year, and a second dose at four to seven years is recommended in most countries. Most developing countries have adopted the WHO EPI to make vaccination accessible to every infant in the world (5). Routine immunizations that are recommended by the EPI schedule include BCG at birth; DTP and oral poliomyelitis vaccines at 6, 10, and 14 weeks; and HB vaccine at 6, 10, and 14 weeks, or at birth and 6 and 14 weeks in regions where perinatal transmission occurs frequently. Recent guidance from WHO has extended EPI to include implementation of universal use of Hib vaccine. Combination vaccines including DTP combined with HB or Hib are given according to the same regimen as DTP. The EPI focuses primarily at protecting infants against vaccine-targeted diseases as early as possible in the first year of life; booster immunizations are considered when high vaccination coverage for primary immunization has been achieved and when economical conditions and health care provision allow it. One dose of measles vaccine is given at nine months, and, in endemic regions, yellow fever is offered at the same time as measles vaccine. The WHO mentions that all children should be offered a second opportunity for measles vaccination. Where recommended, Japanese encephalitis vaccine is usually given in the second year of life, concomitantly with other vaccines administered at the same time. The great diversity in immunization schedules that exists in the industrialized countries complicates the overall clinical development of a new vaccine, resulting in the need for additional studies that will further delay its introduction. The protective efficacy of a new vaccine is classically determined in a double blind, randomized placebo controlled clinical trial, which represents the ideal design for scientifically valid conclusions. Such trials usually require a large sample size and a prolonged follow-up of the study population resulting in huge logistical and financial constraints. Once vaccine efficacy is demonstrated, it is no longer ethical to employ a placebo group in further controlled efficacy trials to evaluate different immunization schedules. Using an immunological correlate of protection, bridging studies allow the extrapolation of efficacy data to other schedules of immunization than that used in the

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Table 1 The DTP Immunization Schedules . . .//. . .

Week of age 6 Austria Australia Belgium Bulgaria Canada Croatia Cyprus Czech Republic Denmark Estonia Finland France Germany Greece Hungary Iceland Ireland Italy Japan Latvia Lithuania Luxembourg Malta The Netherlands New Zealand Norway Poland Portugal Romania Slovakia Slovenia Spain Sweden Switzerland Turkey United Kingdom United States

8

10 12 14 16 18 20 22 24 26 .

. . . .

. . . .

. . . . . .

.

. . . . . . . . . . . . .

. . . . .

. . . . . .

.

.

. . .

. . . . .

. . . . . . . . .

11 12 13 14 15 16 17 18 19 20 21 22 24

.

. .

.

.

. .

. .

. .

.

.

. . . . . .

.

.

. .

.

. .

. . . . .

.

Months of age

. . .

.

. .

. .

.

.

.

. . . . . .

pivotal efficacy trial. It is generally accepted that not all variations of infant immunization schedules need to be evaluated. Indeed, initiating the primary series at an early age and administering it with a short interval between doses are considered conditions that are the most demanding for the immune system of an infant. Demonstration that an accelerated primary series such as two, three, and four months of age (or the even more immunologically demanding EPI schedule) induces an immune response that is non inferior to that used in the original efficacy trial allows extrapolation to less demanding three-dose infant series, that is, three, four, and five months or two, four, and six months (6). By contrast, it will not be possible to project data following vaccine administration at two, four, and six months to either an accelerated schedule or a two-dose primary series. Consequently, several studies are usually needed to permit the use of a new vaccine through the diversity of immunization schedules. This represents a substantial increase in development costs and, more importantly, further delays in making a new vaccine available to all infants. In addition, when vaccines are introduced in different regimens in a particular region of the world, the post-marketing evaluation of vaccination is likely to be more complicated, vaccine

.

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.

. .

.

.

.

.

.

.

.

.

.

. . . . .

. .

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.

.

. . .

effectiveness is more difficult to assess and the interpretation of pharmacovigilance data more complex due to the smaller population available for each regime.

Optimizing Immunization Schedules As discussed above, huge variations exist in immunization schedules around the world, and particularly in wealthy nations. This has largely arisen because of historical reasons and local policy rather than evidence that one schedule provides better protection for children than another. Current understanding of infectious disease control suggests that widespread vaccine coverage, adequate immunogencity, and the presence of herd immunity are likely as important as the timing of the first and subsequent doses of vaccine.

EPIDEMIOLOGY OF DISEASES AND AGE OF IMMUNIZATION In designing an optimal immunization schedule, the epidemiology of the disease under consideration and the age of acquisition, colonization and transmission of the organism are all key variables. For example, in the case of serogroup MenC

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disease, immunization should begin in early infancy, since the highest rates of disease occur among infants and toddlers between the ages of six months to two years. This vaccine approach is based on disease burden and not carriage of the organism, since the highest carriage rates of MenC are among teenagers and young adults and are low in young children (7). Since the introduction of universal MenC vaccination in the United Kingdom, it has become clear that the duration of vaccine induced immunity is short-lived when given in the first 6 months of life (8), with direct protection from vaccine becoming negligible by one year after immunization (9). However, the number of vaccine failures remains very low, presumably because of the high level of population immunity in adolescents and young adults induced by the mass MenC catch up vaccination campaign in 1999–2000. Similar levels of population protection have been achieved without infant immunization, but with a catch up campaign and routine immunization starting at 12 months in several other countries (8). It remains unclear whether a policy of MenC immunization solely in teenage and early adult years, the presumed primary reservoir of the organism, would have the same impact on disease rates as seen with infant immunization programs alone. Another advantage of later immunization is improved immunogenicity of conjugate vaccines, such that fewer doses are needed to provide a similar level of individual protection. For both Streptococcus pneumoniae and Haemophilus influenzae type b, most childhood cases are in the infant and toddler population. In addition, young children are apparently responsible for transmission, particularly with Streptococcus pneumoniae, since there has been a marked decline in pneumococcal disease in older age groups since introduction of infant and toddler immunization with this conjugate vaccine (10). Infant immunization with or without a booster dose in the second year of life had a huge impact on Hib disease. However, in the case of Hib, school age children remain a significant reservoir for carriage in the United Kingdom (Oh et al., unpublished observations) before there was a booster in the second year of life (introduced in September 2006), allowing disease to resurface when vaccine immunity waned (11). To sustain herd immunity, careful monitoring of population immunity is critical. Successful immunization of young children has the potential to reduce circulation of the organism in the population allowing more rapid waning of immunity in other age groups through loss of natural boosting and the possibility of a future resurgence of disease. The age of acquisition and intense transmission of these organisms almost certainly varies in different populations. For example, data from a number of developing countries show very high levels of pneumococcal carriage in the first few months of life (12), whereas first acquisition tends to be somewhat later in developed countries. Thus, since disease occurs soon after acquisition (13), higher rates of disease occur in younger infants in developing countries. Again there is a trade off between the immunogenicity of the vaccines (better in the second year of life and beyond), the rise in the number of cases as maternal antibody wanes in the first year of life, and the age of acquisition of disease. The marked decline in neonatal tetanus in developing countries has been attributed to both improved hygienic practices at the time of birth and to transplacental transfer of protective levels of tetanus antibody to the infant resulting from maternal immunization (14). Levels of circulating

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maternal antibody prevent disease resulting from inoculation of toxin producing Clostridium tetani through the epithelium around the time of birth (15). Outside of the neonatal period, the highest rates of tetanus seen in settings without routine immunization are among older children and young adults, particularly males, who are at greatest risk of receiving a contaminated wound (16). Although tetanus immunization is one of the cornerstones of most immunization programs, there is thus less urgency to immunize against tetanus in the first few months of life in settings where high maternal antibody and optimal neonatal hygiene are present. However, to prevent all tetanus disease, individual protection is required since maternal antibody wanes and exposure may still occur in the childhood years (15). Pertussis is a major cause of morbidity and mortality in regions without an immunization program, with disease commonly seen in the unvaccinated individuals. It also continues to be a problem in countries with universal immunization programs where severe cases with deaths are seen in infants less than two months of age, who are too young to have received vaccine (17). Spread of the organism to the vulnerable infant is most often attributed to parents and siblings (18,19). The observation that adults are important disease reservoirs for infant pertussis appears to be the key to improved pertussis immunization strategies with adult immunization likely to prevent transmission to infants. Emphasis on high antibody levels by the end of the first year of life and sustaining protection through childhood and parenthood appear to be important. Transplacental transfer of maternal antibody appears to confer little protection against disease for the newborn, presumably because of the low levels of pertussis antibody that is transferred and because it wanes rapidly (20). In addition, high maternal antibody levels may impair subsequent infant immune responses to whole cell pertussis vaccines, but not to acellular vaccines (21). Adult immunization also has the potential to provide neonatal protection by ‘‘cocooning’’ the infant with immunized contacts and by raising the level of antibody in pregnancy. Furthermore, transmission of the organism continues in older age groups among whom vaccine-induced antibody has waned, despite the use of booster doses of vaccine. To prevent disease in all ages and serious morbidity and death in early infancy, sustained protection throughout the population can only be produced by repeated booster doses of vaccine. Certainly, inclusion of doses in the second decade of life, as is now recommended in a number of countries, should reduce transmission (22). Consideration of further doses in adulthood, including pregnancy, to reduce household transmission may still be necessary. The success of measles immunization in the Americas has proven that high uptake of vaccine can effectively block transmission of this virus and that there is the potential for global control or elimination of the disease (23). Since the highest rates of death from measles are in early childhood, it is important to provide individual protection as early as possible in communities with ongoing transmission. However, lingering maternal antibody inhibits vaccine virus replication and vaccine-induced immunity is attenuated if immunization occurs in its presence (24). In populations with high vaccine uptake and good control of measles, a delay in vaccination until the age of 12 to 15 months improves the immune response since maternal antibody has disappeared. With this approach, the number of susceptible children is low with little ongoing transmission

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within the community. However, recent experience from the United Kingdom where uptake of measles immunization fell in the late 1990s and early 2000s, resulting in outbreaks of disease, provides clear evidence for the need for sustained high vaccination coverage.

IMMUNOLOGICAL DEVELOPMENT For most antigens immune responses are better in older children and adults than in infants. One of the reasons is interference in the immune response by the transplacental transfer of maternal antibody. Maternal antibody has been shown to have an adverse effect on the immune response of infants to live viral vaccines as mentioned earlier, but also on nonreplicating antigens. In a recent trial of the seven-valent pneumococcal conjugate vaccine in American Indian infants, elevated maternal antibody reduced humoral responses following primary immunization (25). Also Booy et al. found that a two-, three-, fourmonth vaccination schedule for pertussis, diphtheria, and tetanus demonstrated greater interference from maternal antibody than a three-, five-, nine-month schedule (26). Similarly, there is evidence that the magnitude of the rise in antibody after booster doses of the serogroup MenC conjugate vaccine is reduced among those who have the highest pre- booster antibody titers (unpublished observations). Another consideration is the immaturity of the immune system in young children, particularly in their responses to polysaccharide antigens. Plain polysaccharide vaccines in adults are conventionally thought to be T-cell independent type 2 (TI-2) antigens that induce a short-lived rise in serum antibody and do not elicit immune memory. In children under the age of two, such TI-2 responses are absent because the marginal zone of the spleen is immature, resulting in an unsuitable environment for activation of marginal zone B (MZB) cells, the primary cell believed to be involved in plain polysaccharide responses (27). Since the highest rates of disease caused by polysaccharide encapsulated bacteria occur in the under two-year age group, plain polysaccharide vaccines have not been used to prevent clinical infections caused by S. pneumoniae, Neisseria meningitidis, or H. influenzae type b in that age group. However, plain polysaccharides can be made immunogenic in infants by conjugation to a protein carrier that recruits CD4þ T-helper (Th,) cells to provide signals for differentiation of naı¨ve B-cells into plasma cells and memory B-cells (28). This induction of a T-cell dependent (TD) response (29) stimulates germinal centre formation in infants. Nevertheless, the immune response to the conjugate vaccines is still lower in the first year of life than at older ages, even after two or three doses. This explains the immunization schedule for serogroup MenC conjugate vaccine where only one dose is needed after 12 months of age, but two to three doses are needed for individuals less than one year of age. A further issue that is particularly relevant for vaccines administered in early childhood is duration of protection. Infants immunized at two, four, and six months of age with a pneumococcal conjugate vaccine generate IgG antibody responses (30), but the serum antibody wanes rapidly, with some serotype specific antibody levels falling below the protective threshold within a matter of months (31,32). The persistence of immunological memory, which has been readily demonstrated by administration of challenge doses of polysaccharide or conjugate vaccine (inducing a rapid rise in antibody)

might be expected to provide continuing protection. However, recent evidence suggests that the mucosal acquisition of encapsulated bacteria, such as the meningococcus, leads to invasive disease more quickly than the four to six days required for antibody to rise as a result of immunological memory. Similarly, antibody wanes rapidly after immunization with other glycoconjugate vaccines in early infancy such as Hib (33) and serogroup C N. meningitidis glycoconjugate vaccine (MenC) (8), with a corresponding loss of vaccine effectiveness (9,34). This supports the concept that immunological memory is not sufficient for protection against these organisms. This failure of persistence of IgG to capsular polysaccharides after immunization in infancy may be overcome by administration of a booster dose of a conjugate vaccine at 12 to 15 months of age, resulting in a marked rise in IgG antibody levels (35–37). However, it is not yet clear whether there is a sustained antibody response after boosting in the second year of life. This issue is of particular importance in the case of serogroup MenC vaccines, where there is a rise in disease rates again in teenage and early adulthood. Infant immunization has to provide protection 15 to 20 years later if no further boosters are offered. In contrast, it appears that immunization of teenagers induces much more sustained antibody levels, at least after serogroup MenC vaccine (38). Continued assessment will be needed to determine whether additional doses of vaccine are required when children vaccinated in infancy reach adolescence. Although sustaining high levels of antibody seems to be a concern for immunization programs that seek to prevent disease caused by encapsulated bacteria that colonize mucosal surfaces, this is less of a problem for several protein-based vaccines. For example, HB vaccine is highly immunogenic in most individuals, and although antibody wanes over time, protection is sustained, as long as there is an initial immune response (39). This is likely explained by the fact that HB has a longer incubation period, and immunological memory is sufficiently quick to induce protection after exposure.

COMPARING SCHEDULES AND NUMBERS OF DOSES There are relatively few studies that have directly compared the immunogenicity of the same vaccine delivered with different schedules. Taranger et al. compared a two, four, and six-month primary schedule with a booster at 15 months, and a primary schedule at 3, 5, 12 months for diphtheria, tetanus, pertussis, polio, and Hib (40). They concluded that immunogenicity is improved with more doses and greater spacing between doses. In the United Kingdom, the routine DTP immunization schedule was changed from a three-, five-, nine-month regime to two, three, four months in the early 1990s, with the intention of reducing pertussis morbidity and decreasing adverse events associated with vaccine administration. However, Booy et al. found that the two-, three-, four-month schedule was not as immunogenic as the previous U.K. schedule of three, five, nine months for tetanus and diphtheria (26). Similarly, Giammanco et al. compared a DTaP-HepB combination vaccine delivered in either a 2, 4, 6 or a 3, 5, 11 schedule and found that the latter one induced higher antibody levels for several of the components (41). However, like Taranger et al., they also found that the antibody levels measured at seven or six months of age (1 month after the primary series of 2, 4, 6 months or 3, 5 months, respectively) were higher after the three-dose schedule. Carlsson

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et al. demonstrated that a DTaP/IPV/Hib combination vaccine performed well in both: 2-, 4-, 6-, 13-month and 3-, 5-, 12-month schedules (42). Although the three dose primary schedule at two, four, and six months of age induced higher antibody levels than did the three-, five-month schedule, they found no differences in the proportions in each group who had an antibody concentration above the presumed protective threshold for diphtheria, tetanus, Hib, polio, and pertussis. In addition, persistence of antibody was similar between the two groups at follow-up at age five years (43). However, it is noteworthy that there are no data available to assess whether lower antibody response responses seen after the two-dose primary infant series were associated with more reported cases of vaccine-preventable diseases in countries using a three dose 3-, 5-, 11/12-month immunization schedule compared to countries using a four dose 2-, 4-, 6-, 12-month schedule. Controversy exists in the literature as to whether two or three doses of some vaccines are optimal. Some investigators have reported that two doses of pertussis vaccine are not as immunogenic as three doses in a two-, four-, and six-schedule (44) but others have reported comparable antibody responses to pertussis (45), pneumococcal conjugate vaccine (46), meningococcal conjugate vaccine (47), and Hib conjugate vaccines (48–50) after two or three doses of vaccine. In contrast, Funkhouser et al. examined the effect of delaying pertussis immunization until 8, 10, and 12 months instead of the recommended 2-, 4-, 6-month schedule in the United States, and noted that the delay would result in an increase in overall vaccine reactions and an additional 636 cases of pertussis (51). Several authors have found lower responses to two doses of pneumococcal conjugate than to three doses (52) in contrast to the favorable comparison study mentioned above (46). Lower antigen content of the doses of various vaccines are generally less immunogenic than full doses, including pertussis (53) and Hib (54). However, for some antigens, lower doses, even if somewhat less immunogenic than recommended, do not appear to compromise protection and might be administered at a reduced cost in resource-poor settings, for example, Hib (54,55). However, these antigens should be administered at the recommended dose in a particular jurisdiction, as the vaccine indications take account of local epidemiology and the potential for interactions with other vaccines in the local schedule. In general, these examples show that starting immunization schedules later, spacing the doses further apart, using higher doses, and administering more doses, all improve immunogenicity. Conversely, adherence to schedules is improved when the vaccine doses are given close together, when an accelerated schedule is used, as has been shown for HB, and when vaccines are administered in combination (56,57). However, it is encouraging to note that there are no data to suggest that less immunogenic regimens significantly compromise population immunity, as long as boosters are used, although this has not been systematically examined.

COMBINATION VACCINES A combination vaccine consists of two or more immunogens physically combined and administered at the same time in the same anatomic site. Combination vaccines have been developed in an effort to deliver multiple vaccine antigens in a single injection. This will facilitate fewer patient visits and injections, decrease parental anxiety and child discomfort, increase

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compliance with the immunization schedule, reduce storage of numerous different vaccine vials, reduce vaccine errors, and decrease administration costs. Several recent studies have highlighted that combination vaccines improve vaccine delivery and reduce the time to successful completion of vaccine series. Happe et al. demonstrated a highly statistically significant reduction in the time to completion of the primary immunization series in children receiving combination vaccines (58). Marshall et al. evaluated the time to completion of the primary immunization series in the Georgia Medicaid population and found significant reductions in completion time when combination vaccine administration was compared with separate injections (59). Kalies et al. evaluated the use of combination vaccines in Germany and found that the proportion of ‘‘on time’’ vaccinations was greatest for children receiving six valent vaccine combinations when compared with single vaccinations or combinations containing lesser numbers of antigens (57). However, the simple combination of multiple vaccine antigens into one syringe is not without complexities. Combining one vaccine antigen with another may significantly alter the immunogenicity of the individual vaccine antigens as will be described in the subsequent section. For purposes of clarity in the ensuing discussion of combination vaccines, a sign (þ) will be used to indicate vaccines administered simultaneously but at separate anatomic sites, and a virgule (/) will indicate combined vaccines. Pediatric combination vaccines presently licensed or under development are listed in Table 2. Although many combination vaccines have been used routinely for decades, many have been introduced into the routine infant vaccination schedules in the developed countries over the past decade including conjugate Hib, conjugate multivalent pneumococcal vaccine, conjugate serogroup MenC vaccine, trivalent IPV, V, (HB), and HA vaccine. A consequence of the successful development and deployment of new pediatric vaccines is that the number of injections a child must receive in the first 18 months of life has markedly increased. Some parents have voiced concern about the need for multiple vaccines, with one quarter of the parents in the United States believing that their children’s immune systems are weakened by too many vaccines (60). This has resulted in some parents choosing to forego vaccines or to request extra visits to complete the immunization series. Combining vaccines into one syringe may result in a combination product with enhanced reactogenicity or diminished immunogenicity or efficacy, compared to the separately administered vaccines. There are several mechanisms proposed to explain how one vaccine might interfere with the immunogenicity of another when mixed together. Physical or chemical interaction between components of the vaccines might alter the conformation of the necessary epitope. As an example, studies of the combined IPV and diphtheria toxoid-tetanus toxoidwhole cell pertussis (DTwP) vaccine demonstrated that the preservative thimerosal destroyed the potency of IPV (61). Another example was that the pertussis component of DTwP lost potency over time, due to destruction of antigen without merthiolate stabilizer (62,63). Mixing an adjuvanted vaccine with one that does not contain an adjuvant can cause displacement of one vaccine from its adjuvant, resulting in diminished immune response to that component. Interference between components of live viral vaccines has also occurred. One such instance was the reduced seroconversion to the mumps component of a bivalent mumps-measles vaccine (64). Viral strain interference is thought to be a consequence of competition for

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Table 2 Combination Vaccines Presently Licensed or Under Developmenta,b Combination has been licensed Vaccines combined Td/IPV DT/IPV DTwP/IPV DTwP/Hib

DTwP/Hib/IPV DTwP/HepB DTwP/HepB/Hib DTwP/Hep B/Hib2.5 DTwP/HepB/MnC/ Hib DTwP/Hep B/MnAC/ Hib DTaP/IPV DTaP/Hib DTaP/IPV/Hib DTaP/HepB DTaP/IPV/HepB DTaP/Hib/HepB DTaP/Hib/IPV/HepB DTap/Hib/IPV/HepB/ MNC Tdap/IPV

HepB/Hib HepB/HepA HepA/typhoid MMRV

MnC/Hib

In Europe or Canada

In the United States

SP-MSD, SP-Ca SP-MSD, SP-Ca SP-MSD, SP-Ca SP-MSD, SP-Ca, GSK, Wyeth, Chiron SP-MSD, SP-Ca GSK GSK GSK (licensed in Europe/Hungary)

In other countries

Clinical trials conducted

SP-Fr SP-Ca, SP-Fr, Novartis SP-Ca, SP-Fr GSK GSK

Merck

GSK GSK (clinical trials) SP-MSD, SP-Ca, BL, GSK, SSI SP-Ca, GSK SP-MSD, SP-Ca, GSK, SSI GSK GSK GSK

SP-MSD, GSK (licensed in Europe) SP-MSD GSK SP-MSD; GSK

SP-USc

SP-Ca, SP-Fr, GSK SP-Fr, GSK SP-Ca, GSK

SP-MSD, GSK

GSK GSK GSK GSK, SP-Fr

Merck GSK

GSK

GSK

GSK (licensed in other countries/ Australia)

BL

SP-US, Merck GSK

Merck

GSK (licensed in Europe/UK)

MnCY/Hib PnC/MnC PnC/MnC/Hib

GSK (clinical trials) Wyeth Wyeth

a Products combining only multiple serotypes of a single pathogen are excluded, as are DT, DTP, DTaP, OPV, IPV, and MMR. Only those manufacturers who distribute their products internationally are listed; other manufacturers may produce some products (e.g., DTP/IPV) for local or regional use. Some products represent components derived from, or joint efforts of, more than one manufacturer; in such cases, their principal distributor is shown. b No discrimination is made between products distributed in combined form and those distributed in separate containers, for combination at the time of use. c Licensed for the fourth (booster) dose only. Abbreviations: aP, acellular pertussis vaccine (infant formulation); ap, acellular pertussis vaccine (adolescent/adult formulation); BL, Baxter Laboratories; D, diphtheria toxoid vaccine (infant formulation); d, diphtheria toxoid vaccine (adolescent/adult formulation); GSK, GlaxoSmithKline; HepA, hepatitis A vaccine; HepB, hepatitis B vaccine; Hib, conjugate Haemophilus influenzae type b vaccine; IPV, enhanced inactivated trivalent poliovirus vaccine; MMRV, measles, mumps, rubella, and varicella vaccine; MnC, meningococcal conjugate vaccine (serotype C initially, additional serotypes subsequently); PnC, pneumococcal conjugate vaccine (7-valent initially, 13-valent subsequently); SP-MSD, Sanofi Pasteur MSD; SP, Sanofi Pasteur (CA, Canada; Fr, France; US, United States); SSI, Statens Seruminstitut; T, tetanus toxoid vaccine; WP, whole-cell pertussis vaccine. Source: From Ref. 65.

mucosal receptors or lymphocyte binding sites. It is also possible that the immune responses, such as interferon production, stimulated by one viral vaccine strain may interfere with the replication of another vaccine strain. New combinations of vaccine products therefore must be evaluated with the same methods and rigor as if they were a new vaccine.

REGULATORY ASPECTS OF COMBINATION VACCINES National regulatory agencies are responsible for the licensure of new combination vaccines. The FDA of the United States must follow the Code of Federal Regulation (CFR) with two different codes applying to approval of combination products. One of

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these, 21 CFR 300.50, states that a fixed combination prescription drug must demonstrate that each component makes a contribution to the claimed treatment effects, and that the dosage of each is safe and effective. It is further stipulated in 21 CFR 601.25(d) (4) that safe and effective active components may be combined if each component makes a contribution to the claimed effects, combining does not decrease purity, potency, safety, or effectiveness of the individual component, and when used correctly, provides preventive therapy or treatment. The FDA issued in 1997 a ‘‘Guidance for Industry for The Evaluation of Combination Vaccines for Preventable Diseases: Product, Testing and Clinical Studies’’ for the purpose of assisting industry in the manufacture and testing of combination vaccines. To ensure combination vaccine licensure, the safety, immunogenicity, and efficacy of combination vaccines must be compared to those of the individual components in prospective, randomized, blinded trials. Two general approaches may be used. The fully combined final product (A/B/C/D) may be compared to each of its components (AþBþCþD). A disadvantage of this method is that it requires multiple arms (2n), and the sample sizes are large. It also carries the danger that, if there is enhanced reactogenicity or diminished immunogenicity with the combination, it cannot be determined which component(s) are responsible. Therefore, the evaluation method most commonly used is to proceed in a stepwise manner. If the ultimate goal is to combine four vaccines (A/B/C/D), the initial study compares bivalent A/B to AþB. If no interference is observed with the bivalent vaccine, the next evaluation will be of A/B/C versus A/BþC. If that is also successful, then a final study comparing A/B/CþD versus A/B/C/D would be performed. The optimal study compares protective efficacy of a combination vaccine to its components administered separately. However, this is often not possible since the routine use of the single component vaccine has already dramatically reduced or eliminated the disease under study. A trial that demonstrates efficacy in one country may be used to support licensure in a different country if a bridging study is performed, showing that the immunogenicity of the vaccine component in the second population is similar to that in the original efficacy study. The use of serologic markers of protection is another approach. For example, the presence of neutralizing antibody to polioviruses is often considered proof of immunity to infection. Therefore, if a poliovirus vaccine is combined with another product, one simply must demonstrate that the combination product stimulates neutralizing antibodies to the three poliovirus strains contained in the vaccine to the same extent as the administration of IPV alone. This method is only feasible with diseases for which serologic correlates of protection have been established, such as diphtheria, tetanus, HB and, arguably, Hib, and group MenC and Streptococcus pneumoniae. Unfortunately, immunologic correlates of protection are not recognized (or are not widely accepted) for some pathogens, such as Bordetella pertussis. The statistical approach to evaluation of combination vaccines is to demonstrate non-inferiority (one-sided equivalence) of the components in the combination compared to the components administered separately (66). Immune response endpoints in non-inferiority trials are often geometric mean concentrations or titers of antibody (GMC/GMT) and/or the proportion of children achieving a defined level of antibody.

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EVALUATING SAFETY OF COMBINATION VACCINES As a general rule, systemic adverse events are increased only modestly, if at all, after the combination administration of multiple vaccines compared with administration of the most reactogenic single vaccine alone. Local adverse events are often somewhat more common and more severe at the site of injection of the combination but this increase is usually offset by the reduced number of total injections needed. So far, no combination vaccine has elicited a new type of reaction not previously seen with its components. For a combination vaccine based on well-characterized components, the combination is compared to its components given separately or to another licensed combination for rates and severities of adverse events. Although non-inferiority (or superiority) is desired, a modest increase in minor adverse reactions is often considered acceptable, recognizing that there has been a concomitant decrease in the total number of sites experiencing local reactions.

Combination Vaccines Based on Acellular Pertussis Vaccine The development and licensure of numerous acellular pertussis vaccines in combination with diphtheria and tetanus toxoids (DTaP) over the past decade has represented an important milestone for safe and effective vaccines for young children. It also rapidly stimulated combinations of DTaP with other routine vaccines of infancy, such as Hib, IPV, and HepB. Building on the experience with DTwP combination vaccines, efforts turned first to evaluating combinations of DTaP and conjugate Hib vaccines. However, it was soon found that combining DTaP with Hib tended to reduce, often markedly, the Hib antibody response (46). Some specific examples highlight this problem. Schmitt et al. found that mixing DTaP and Hib vaccines for primary immunization substantially reduced the Hib antibody responses (67). However, they were not concerned by this finding, since the proportion of children achieving ‘‘protective levels’’ were similar. Similarly Kitchin et al. described a trend toward reduced Hib antibody concentration when the Hib vaccine was combined as part of a DTaP/IPV/Hib combination when compared to DTwP-Hib (68). These interferences in the immune responses to Hib slowed the development of combination vaccines based on DTaP/Hib and stimulated development of alternative combinations such as DTaP/IPV, DTaP/HepB, and DTaP/IPV/ HepB. It also prompted research into the clinical relevance of the reduced Hib responses. Hib-containing pentavalent and hexavalent (DTaP/IPV/Hib/HepB) combinations have been licensed in Europe and some other jurisdictions, despite reduced Hib responses. In contrast, attention in North America has focused on DTaP/IPV/HepB and on DTaP/Hib-based combinations built on the Canadian five-component DTaP, which appears not to interfere materially with Hib responses. The clinical relevance of these differences must await continued surveillance in all jurisdictions. Combining IPV with DTaP or with DTaP/Hib has had no consistent effect on antibody responses to the DTaP and IPV components, with few differences achieving statistical significance (69,70). In addition, ongoing surveillance in Sweden has shown continued reductions in pertussis incidence among the vaccinated population, concomitant with the transition from DTaP to DTaP/IPV and DTaP/IPV/Hib (71). Combining HepB with DTaP or with DTaP/IPV generally produces somewhat higher DTaP and polio antibody responses than are achieved

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with the same components given separately on the same schedule (41,72). However, the HepB responses following the combination vaccines typically are lower than those seen with monovalent HepB, not because of interference but because administration schedules for combinations typically are more closely spaced than are schedules for monovalent HepB. The magnitude of HepB antibody responses is directly correlated with the time between doses. Accordingly, HepB responses are lower if the HepB is administered (whether separately or in a combination) at, for example, 2, 4, and 6 months or 3, 4, and 5 months rather than at, for example, 0, 1, and 6 months or 3, 5, and 11 months. A combination vaccine of recombinant HepB vaccine and PRP-OMP conjugate Hib vaccine is licensed in the United States and Europe. A study comparing this combination vaccine and its constituent components administered to infants found no material difference in antibody responses (73). A combination incorporating HepA and HepB antigens is available in the United States, Canada, Europe, and elsewhere in both adult and pediatric formulation. A comparative trial of the combination hepatitis vaccine with its individual components given separately at 0, 1, and 6 months found excellent antibody responses in adults, with 100% of combined-vaccine recipients achieving protective levels of both antibodies before the six-month injection (74). Long-term follow-up studies confirmed that after the combination product, adults remained seroprotected. Finally, combination vaccines to prevent meningitis and invasive bacterial infections with encapsulated bacteria are also under development. Classically, the three most common causes of bacterial meningitis are Hib, meningococcus, and pneumococcus. Effective conjugate vaccines exist for all three agents, and combining these vaccines offers obvious epidemiological synergies. Unfortunately, development of such combinations has been set back by interference between the agents. The first successful combination vaccine, MenC/Hib, was recently licensed by GlaxoSmithKline (GSK) in Europe for use in infants and toddlers. This combination, given in association with DTaP/IPV or DTaP/IPV/ HB at two, three, and four or at two, four, and six months, produced Hib antibody levels that exceeded, and meningococcus serogroup C responses that at least equaled, those seen in comparison groups given separate vaccinations (75) Wyeth Lederle has also conducted clinical trials of PnC/Hib, PnC/ MenC, and PnC/MenC/Hib combinations. A comparison of seven-valent (7v) PnC and HbOC, given combined or separately, found significantly lower antibody concentrations in the combined group for five of the seven pneumococcal serotypes. Hib responses did not differ significantly with respect to the proportions achieving certain defined ‘‘correlates of protection’’ (76). In contrast, a study comparing 7vPnC/MenC/HbOC with the separate administration of HbOC plus either 7vPnC or 9vPnC/MenC found that GMTs and seroresponse rates in the combination vaccine groups met the statistical criteria for noninferiority for the pneumococcal antigens, but the PRP antibody response to the PnC/MenC/HbOC combination was inferior to that of separately administered HbOC (77). This reduced Hib responsiveness, thought possibly to represent antigen overload with the carrier protein, may prevent inclusion of that component in future PnC/ MenC combinations. As mentioned in an earlier section, Kitchin et al. (68) found a reduction in Hib antibody concentration when MenC-CRM vaccine was concomitantly administered (with either aP or wP), as compared to concomitant administration of MenCTT. In contrast, others have shown that MenC-CRM has no effect on Hib immune responses (78). Buttery et al. evaluated a Wyeth

PnC9/MenC combination (all components conjugated to CRM197) versus monovalent CRM197 MenC vaccine, with each group also receiving DTwP, Hib, and OPV (79). MenC serum bactericidal antibody titers, and Hib and diphtheria antibody responses were all lower with the combination. The investigators concluded that these results ‘‘may limit the development of the multiple conjugate bacterial vaccines.’’

MORE VACCINES TO INCORPORATE IN THE SCHEDULE To control infectious diseases in the future, there are a large number of antigens that can be added to immunization schedules beyond the established set of antigens. There are some which are relevant only to specific regions (such as Japanese encephalitis vaccine) but an increasing number with global relevance that are not yet universally adopted (e.g., several conjugate vaccines, HB, oral rotavirus vaccine, human papilloma virus vaccine). For parents and vaccine providers, the increasing number of needles and visits that are required for injecting these antigens in infancy is a substantial obstacle. However, early immunization may be important in maintaining high coverage since attendance at immunization clinics is more difficult once the child is older. Improving population acceptance of immunization and developing immunization services, particularly in the second decade of life, is a priority for improving immunization schedules. Several vaccines in development could be incorporated in the primary immunization schedule in the foreseeable future, including new generation pneumococcal vaccines, influenza vaccines, and vaccines for serogroup B meningococci (MenB). As an example, although several MenB vaccines are currently in clinical trials, those that have been tested so far have shown poor immunogenicity in early infancy (80). If a MenB vaccine is licensed and shown to induce herd immunity, it may be used in a catch-up campaign at implementation and incorporated into teenage immunization schedules to sustain herd immunity among young adults. Consideration of future immunization programs should include the possibility of adding further vaccines, such as MenB, and highlight the need to create space or develop new combinations. If new MenB vaccines are poorly immunogenic in early infancy, then a schedule starting at 6 months of age may provide similar immune responses to one starting at 12 months (81), though this may be too late to prevent some cases.

A PROPOSED SCHEDULE FOR IMMUNIZATION We have outlined a possible immunization schedule that incorporates new antigens and builds on research described above to balance rates of disease in early childhood with epidemiology of the organism and maturity of the immune system (Table 3). Perhaps, the schedule that best fits with the principles discussed above is a Scandinavian schedule of 3, 5, 12 months for routine immunizations. This schedule optimizes immunogenicity by starting later and provides space for addition of new antigens, and, importantly, local disease surveillance in this region does not indicate any compromise of population protection. However, there are no studies that have demonstrated definitively that one schedule is better or worse than those that are currently available. Each new schedule needs evaluation, especially for interactions between vaccines and must take account of local issues such as vaccine uptake and disease epidemiology.

Chapter 41: Rationalizing Childhood Immunization Programs

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Table 3 Suggested Immunization Schedule that Minimizes Numbers of Doses and Creates Space for Additional Antigens to be Inserted Vaccine

Notes

Routine schedule DTaP-IPV-HibReduce primary course to 2 doses by Hep B dropping 6 mo dose—could start 1 mo later (at 3 mo) for improved immunogenicity PCV7 or PCV10 Reduce primary course to 2 doses by or PCV13 dropping 6-mo dose once herd immunity established? Oral rotavirus 2 or 3 doses depending on product used MenACYW New vaccines awaited with better immunogenicity for toddlers, may need 2 priming doses with current vaccines. Could combine with MenB? MenB (when MenB vaccines may be less available) immunogenic in early infancy so may need to start course later. Need to add extra vaccine visit creating opportunity for other antigens at new visit. Possible combination with MenACYW to reduce needles at 13-mo visit? If immunogenic, then should be started before 6 mo of age in view of high rate of disease at this age dTaP-IPV Consider additional adult doses to reduce pertussis circulation— perhaps offered to parents at baby vaccine clinics. MMRV Possible later boosters to reduce risk of shingles HPV 3 doses. No data on need for later boosters Influenza Seasonal influenza vaccine for infants facing their first winter over 6 mo of age—2 doses Hepatitis A/B Schedule for countries that do not have Hep B in the infant schedule— 3 doses Number of needles per visit Special situations Hepatitis B For high risk infants BCG For high risk infants

1 Birth mo

2 mo

3 mo

4 mo

5 mo

6 mo

8 mo

12 13/14 4 mo mo yr

x

x

x

x

x

x

x

x

11 yr

13 to – 15 yr

x

x

x

x

X

x

X

x

x

X

x xxx

xx xxx 2

x x

x

x

2

1þ

1þ

2

2þ

2

2

2þ

x

Note that physicians should always adhere to local licensed schedules. The suggested schedules in this table are hypothetical and should be formally studied prior to implementation. Abbreviations: D, diphtheria; T, tetanus; aP, acellular pertussis vaccine; IPV, inactivated polio vaccine; Hib, Haemophilus influenzae type b vaccine; PCV, pneumococcal conjugate vaccine; MenACYW, meningococcal vaccine containing serogroups ACYW; MenB, meningococcal vaccine containing serogroup B; MMRV, measles, mumps, rubella and varicella vaccine; HPV, human papilloma virus vaccine.

It is likely that schedules that have more than two injections at each visit will not be popular with parents or physicians. There is therefore a balance between (i) reduced coverage and delayed immunization if there are too many needles and (ii) the reduced immunogenicity of some of the combination vaccines. It is therefore important that further development of combination vaccines continues, or preferably new delivery systems that are more acceptable are developed, so that the number of needles is reduced. Herd immunity is key to reducing the number of doses in the first year of life. For example, if toddler, adolescent, and adult immunization

reduces pertussis transmission, then the concerns about the merits of two or three priming doses may be less important, making space for new antigens. Similarly, if there is no MenC, Hib, or pneumococcal transmission, then slightly lower population antibody levels following two priming doses in infancy may sustain sufficient population immunity until booster doses in the second year of life. This will be a particular issue for MenB vaccines (if those currently in trials prove to be sufficiently immunogenic) as the available data indicates that three to four doses are required to produce adequate antibody levels. Adding MenB to the schedule will result in a

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substantial number of additional injections and visits, unless combinations of antigens, such as MenABCYW could be designed. Countries that have not yet added HB vaccine to their schedules have the possibility of adding this antigen at the time of HPV vaccine introduction for adolescents, and these two vaccines could perhaps be combined to simplify the regime, possibly with HA. While immunizers should always adhere to local recommendations, future immunization programs should seek to maintain broad protection of the population against a wider range of vaccine-preventable infectious diseases while minimizing the number of vaccination visits, reducing the number of doses and needles administered to infants in the program, and improving long-term immunogenicity.

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42 Meningococcal Conjugate and Protein-Based Vaccines Andrew J. Pollard and E. Richard Moxon Oxford Vaccine Group, Department of Paediatrics, University of Oxford, Children’s Hospital, Oxford, U.K.

Rino Rappuoli Novartis Vaccines and Diagnostics, S.R.L., Siena, Italy

INTRODUCTION Neisseria meningitidis remains one of the leading infectious causes of death in childhood in many industrialized countries and a cause of devastating epidemics in nonindustrialized nations. Some 500,000 cases of endemic meningococcal infection are thought to occur annually worldwide (1), with the greatest burden of disease in Africa and Asia. Of great public health importance, meningococci are the cause of cyclic epidemic meningitis in Africa (2) and Asia (3), and occasional outbreaks have been associated with population movements and overcrowding in other regions over the past half century (4). Currently, five serogroups of meningococci, A, B, C, Y, and W135, defined by the biochemistry of their polysaccharide (PS) capsule, are responsible for almost all meningococcal disease, although the overall proportions of cases caused by each serogroup vary widely around the globe. An epidemic of serogroup X meningococcal disease, with many thousands of cases, in Niger in 2006 (5) highlights the potential for other serogroups, usually associated with very low rates of sporadic disease, to be important contributors to the global disease burden. In industrialized nations serogroup B meningococci cause 30% to 90% of cases of sporadic meningococcal disease (6–8) and have been responsible for pockets of persistently increased rates of disease (9–11). Serogroup C meningococci are particularly associated with small outbreaks of disease among teenagers and young adults and sporadic disease in individuals of other ages (7). Serogroup Y disease is uncommon in the United Kingdom, but accounts for up to 30% of cases in the United States (7) and rates of Y-disease may be on the increase in parts of Canada too (8). Occasional sporadic disease caused by W135 meningococci had been largely ignored until a recent large outbreak among pilgrims to the Hajj in 2000 (12) and subsequent epidemics in the meningitis belt of Africa over the following few years (13). Although the epidemiological characteristics of disease caused by each serogroup are intriguingly different, the clinical features of invasive disease are mostly indistinguishable. The shifting epidemiological patterns of meningococcal carriage and disease are poorly understood and the lack of predictability concerning the future spatial and temporal distribution of this pathogen must be taken into account as vaccines are developed. Nonetheless, several new vaccine initiatives provide the possibility of a major reduction in the global burden of this disease during the coming decade.

The contribution of vaccines in the control of meningococcal disease is particularly important because of the rapidity of its onset and the short window of time that may exist between acquisition of the organism and fulminant disease and death. Antibiotics, optimization of early management (14), and specialist intensive care have significantly reduced the mortality from meningococcal septicaemia (15) in specialized centers in industrialized countries. However, the timely, universal availability of such treatment cannot be provided for most of the world, particularly in countries where access to antibiotics and primary health care, let alone sophisticated intensive management, is virtually unavailable on a day-to-day basis. Widespread control of meningococcal disease is an important public health goal, and immunization is the only realistic means to achieve this aim. The capsular PSs of meningococci are the key to its virulence, and the fact that serum antibodies directed to these surface-exposed carbohydrate antigens provide protection against invasive disease has been exploited in the development of vaccines. Indeed, the protective role of antibodies able to promote complement-mediated killing of bacteria [serum bactericidal activity or (SBA)] was demonstrated in the 1960s (16). This information was used to develop and license vaccines composed of purified capsular PSs, which today are available as bivalent vaccines against serogroups A and C or as tetravalent vaccines against serogroups A, C, Y, and W135. Although there is proven efficacy for MenA and C plain PS, PS vaccines are still used only in people at increased risk (for instance military recruits) or in response to outbreaks. They have never been considered for universal vaccination because of several shortcomings that compromise their utility. Specifically, immunization with MenA PS does not confer long-term protection (it is effective for about 6 years) and MenC PS is unsatisfactory as a vaccine in children younger than 2 years as it is not immunogenic and efficacy below 10 years is limited (17). Improved immunogenicity and longer-term protection, especially in infants and young children, can be realized through conjugation of the capsular PS to carrier proteins. Described for the first time in 1992 (18), protein-PS conjugate vaccines (CV) were introduced in the U.K. primary infant schedule in November 1999 against MenC, after many years of careful assessment of their safety and immunogenicity. They had a major impact on disease caused by Serogroup C meningococci in childhood in

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the United Kingdom (Ref. 19, see chap. 45). MenC CVs have also been licensed in other countries around Europe (20) and in Canada (21). Combination A-C CVs have been proven immunogenic in clinical trials performed in infants and children (22) but these bivalent vaccines were not pursued to licensure. A quadrivalent diphtheria toxoid–conjugated meningococcal ACYW CV (MenactraTM, Sanofi Pasteur) was licensed for use in 11- to 55-year-olds in January 2005 in the United States and in 2- to 10-year-olds in May 2006 in Canada. Data concerning one other A, C, Y, and W135 protein-PS CV (conjugated to CRM197) has also been reported (23,24). In infants, there was a poor immune response to the diphtheria-conjugated ACYW CV after a three-dose (2, 4, and 6 months) schedule with protective levels of bactericidal antibody reached against MenC in 54.2%, MenY in 66.7%, MenW135 in 62.5%, and MenA 91.7% (25). However, priming was demonstrated by a response to all serogroups after a PS booster in the second year of life. By contrast, higher seroconversion rates were achieved with the CRM197 CV with protective titers reached against MenC in 84%, Y in 92%, and W-135 in 96% after immunization at two, three, and four months (26). In several studies of toddlers with two doses of the diphtheria-conjugated ACYW vaccine, protective titers of bactericidal antibody were achieved in over 90% of the participants (25,27). A study in children from 2 to 10 years with the diphtheria CV showed that protective titers of serum antibody were elicited in a comparable proportion of subjects as children given the previously licensed plain PS vaccine (28), though protective titers waned over the next few years in most participants (29), as has been described for other CVs. Similarly in adolescents, both the diphtheria CV and plain PS vaccine were immunogenic in a comparative trial, but antibody persistence was better in those receiving the CV (30). Various other ACY and W135 combination vaccines are also in development (other ACYW CVs, Hib-MenCY, and HibMenAC-DTPw-HB) and a Hib-MenC CV has been used in the United Kingdom since 2006 as booster vaccine at 12 months. A monovalent serogroup A CV is under development by the Meningitis Vaccine Project (a partnership between PATH and WHO) to provide a low-cost solution for serogroup A disease in the meningitis belt of Africa (31). These developments provide the potential for global disease control of ACY and W135 meningococcal disease. Progress toward the global control of disease caused by A, C, Y, and W135 must be tempered in view of the current failure to find a solution to the problem of MenB disease. The highest attack rate of meningococcal disease is in children younger than five years and, at this age, 50% of disease is caused by MenB in the United States (199–1996) (7), 39% in Canada (1985–2000) (8), and more than 90% in the United Kingdom (2004) (6). As for serogroups ACY and W135 described above, the PS capsule of MenB is attractive as a vaccine antigen because, by definition, it is shared across this group of meningococci. However, the PS capsule of the serogroup B meningococcus is a homopolymer of sialic acid, chemically identical to PSs found in human tissues, especially fetal brain during development (32). Hence, the Group B capsule is seen by the immune system as a self-antigen, and this may explain in part its poor immunogenicity even after its conjugation to a protein carrier (33). Since this approach proved so successful for other capsular PSs, Jennings et al. pioneered an innovation in which chemical modification of the PS (N-propionylation) retains immunogenic

epitopes. This approach has resulted in the development of a protein-PS CV that elicits functional (bactericidal) antibody in both mice and nonhuman primates (34,35). Some of the antibodies elicited have activity against polysialic acid and therefore have the potential to be autoreactive in humans, although no deleterious effects have been noted in early human trials (P. Fusco, Baxter, 2001, personal communication). Nonetheless, there is a strong sense that the strategy of PS-protein conjugation is not attractive to vaccine developers who anticipate ethical and regulatory difficulties that may be difficult, if not impossible to resolve in taking forward these vaccines as commercially viable propositions. However, other antibodies that arise after immunization with a conjugate N-propionylated serogroup B PS vaccine do not cross-react with human tissues. A derivative approach that might avoid the cross-reactivity issue is to use molecular mimetics of non-autoreactive epitopes as potentially safe serogroup B vaccine antigens (36). The problems encountered in the development of MenB PS-based vaccines has resulted in consideration of a variety of alternative candidates, notably outer membrane vesicle (OMV) vaccines, recombinant outer membrane (lipo)protein vaccines, and lipopolysaccharide (LPS) vaccines. However, while so far evaluation of the potential utility of these alternative MenB vaccine candidates has been difficult because of the lack of an accepted laboratory surrogate of protection at a recent consensus meeting held in Atlanta (37), it was concluded that serum bactericidal antibodies (dilutions of serum that can kill meningococci in the laboratory in the presence of complement) are ‘‘a good surrogate for predicting the effectiveness of a meningococcal group B vaccine’’ and that ‘‘immunogenicity based on functional SBA activity will be the primary end point for evaluating vaccines’’ (16), as clearly described for serogroup C meningococci (38). It is not clear whether other assays, such as opsonophagocytosis or protection in animal models, might better reflect protection of humans against MenB disease. An important question is the degree of serological cross-protection required before a vaccine could be licensed and used widely. It is therefore noteworthy that the MenC vaccine when introduced in various regions reduced the burden of meningococcal disease by 30% to 50%, and this might be a reasonable target for a first-generation MenB vaccine.

OUTER MEMBRANE PROTEIN VACCINES The outer membrane proteins (OMPs) of MenB have been extensively studied as potential vaccine constituents since the 1970s. A drawback to their candidacy as vaccines is that these proteins tend to be highly variable not only among different MenB isolates but also within clonal populations of the same strain. As a consequence, any OMP from a single strain is unlikely to provide cross-protection to all other MenB strains. Furthermore, the antigenic regions of many of these protein structures evolve rapidly within bacterial populations because of the natural selection on carriage strains, especially through the acquired host immune clearance mechanisms mediated by local and systemic B cells. However, despite these reservations, there is good evidence for conservation of OMPs within clonal complexes, so that a small number of variants of certain OMPs are stably associated with a particular lineage over long periods of time and in different geographic locations (39,40), providing the possibility that a relatively small number of proteins, particularly in combinations of different proteins, might constitute a cross-protective vaccine.

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Table 1 Summary of the Efficacy Trials Against Meningococcus B Performed with Outer Membrane Vesicle Vaccines Trial

Vaccine

Doses

Efficacy adults

Norway 1988–1991

NIPH 15:P1.7,16

2 doses

Cuba 1987–1989 Brazil 1990–1991

Finlay 4:P1.19,15 Finlay 4:P1.19,15

2 doses 2 doses

87% 57% 83% 74%

Chile (Iquique) 1987–1989

Cuban type 15: P1.7b,3

2 doses

70% (30 mo) (5–21 yr)

(10 mo) (29 mo) (13–16 yr) (10–14 yr) (>48 mo)

Efficacy children

Efficacy infants

/

/

/ 47% (24– 47 mo) /

/

Efficacy total Reference 26

37% (<24 mo)

54%

23 27

23% (30 mo) (1–4 yr)

51%

25

Table 2 Strain Specificity of the Immunoresponse in Infants and Children after Immunization with OMV Vaccines Immunogenicity Norwegian vaccine

Immunogenicity Cuban vaccine

Test strain

Infants

Children

Adults

Infants

Children

Adults

Chile Cuban Norwegian

12 2 98

35 24 98

60 46 96

10 90 31

31 78 41

37 67 56

Note: Comparison of bactericidal responses against homologous and heterologous strains induced in infants, children, and adults in a clinical trial in Iquique (Chile) using the Cuban and Norwegian OMV vaccines. As shown, three doses of OMV vaccines induce good bactericidal titers against the homologous strain at all ages. No or low titers against heterologous strain in infants and children, respectively. Abbreviation: OMV, outer membrane vesicle.

Table 3 Bactericidal Activity Against a Panel of Different Strains Induced by Three Prototype Antigens Against Meningococcus B . Strain OMP . OMV . Non-OMP

B

B

B

B

B

B

A

C

C

2996 16000 65000 65000

BZ232 2048 8000 512

1000 – – 4000

MC58 – 2048 8000

NGH38 – – 32000

394/98 – – 4000

F612 – 32000 8000

C11 – – 1024

BZ133 – – 16000

OMP is a purified membrane protein with a predicted typical b-barrel structure spanning the outer membrane. OMV is a typical membrane vesicle vaccine produced from strains 2996. Non-OMP is a new surface-exposed protein discovered with the genomic approach. Abbreviations: OMP, outer membrane protein; OMV, outer membrane vesicle.

The earliest OMP vaccines comprised insoluble aggregates of OMPs that were poorly immunogenic in humans (41). Purified OMPs noncovalently complexed to meningococcal C-PS were tested in a trial in Chile and resulted in substantial protection in older children and young adults (70%), but were poorly protective in children younger than five years (42). To present OMPs in an appropriate conformation and thus improve immunogenicity, OMP vaccines were produced in spheres of bacterial outer membrane, known as OMVs (42). These OMV vaccines have now been evaluated in large-scale trials (Table 1) and, using two doses, have resulted in substantial protection in older children (42–44), but not children younger than four years (42,43,45,46). Furthermore, bactericidal antibody tends to be directed to the serogroup B meningococcal ‘‘type strain’’ included in the vaccine (Table 2) (47,48). The major target of protective immunity in these OMV vaccines is PorA, a porin that is the basis of the serosubtyping system of meningococci and therefore known to be antigenically variable. The role of PorA as a target for protective antibodies, the problem of its variability and the lack of cross-strain protection have stimulated research culminating in secondgeneration OMV vaccines. This approach used genetic techniques so that six different PorA proteins, expressed in two engineered strains, were included (Table 3). Phase II trials in various age groups have demonstrated variable immunogenic-

ity to the PorA types and the need for multiple doses (49). Furthermore, mutations in the genes encoding this protein are common and result in evasion of complement-mediated killing of the organism, further complicating the success of this approach (50). Nonetheless, the observation that some protection and induction of bactericidal antibody directed against the vaccine strain is induced with monovalent OMV vaccines suggests that these vaccines may be of use in outbreaks of serogroup B disease caused by a single clone of bacteria. For example, over more than a decade, the rates of MenB disease in New Zealand rose to 4 to 20 times higher than other industrialized countries (9). In July 2001, the Ministry of Health in New Zealand announced plans for further development of a monovalent OMV vaccine with Novartis Vaccines (formerly Chiron Corporation) in conjunction with the National Institute for Public Health (NIPH), Norway, in the hope of halting serogroup B disease (51). A safety and immunogenicity study of the tailor-made B:4:P1.7b,4 New Zealand serogroup B OMV vaccine (MeNZB, Novartis vaccines, Siena, Italy) found the vaccine to be safe and immunogenic in adults, with up to 100% responding with a fourfold rise in bactericidal antibody level after three doses, with higher response rates associated with the highest dose (52). Reactogenicity rates were similar to those in a study arm

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receiving the Norwegian OMV vaccine that had been used in trials in Norway previously. After three doses of MeNZB, 75% of infants 6 to 8 months and toddlers 16 to 24 months showed a fourfold rise in bactericidal antibody, but four doses were required before similar responses were observed in younger infants (53). Fever (>388C) was observed in up to 24% of 6- to –10-week-old infants, with the highest rates after the second dose. Persistence of protective titers of serum bactericidal antibody was observed in less than one-third of the infants and toddlers at 5 to 16 months after three doses, but significant rises were seen in almost all individuals after a fourth dose (53). As had been observed with previous OMV studies, the bactericidal antibody was directed at the PorA protein, specifically in this case the VR2 P1.4 epitope (54). Infant responses were dominated by IgG1 and IgG3 (55). On the basis of these data, 1 million individuals were immunized in New Zealand to control the epidemic. A preliminary safety review of the campaign was favorable (56), effectiveness data are not yet available; however, the reported cases have decreased from 450 to 650 per year during the period 2000 to 2004, before the beginning of the vaccination campaign to 160 in 2006 (57). A bivalent vaccine consisting of OMVs from the Finlay Institute B:4:P1.19,15 strain and the New Zealand B:4:P1.7-2,4 strain with aluminium hydroxide was studied in a three-dose schedule in healthy adolescents (58). A fourfold rise in antibody was identified in 51% of volunteers against P1.19,15 strains and in 66% against P1.7-2.4 strains. In total, 28% to 46% of volunteers had a fourfold rise in antibody against three heterologous strains, implying the presence of cross-protective epitopes in the vaccine. The presence of cross-protective responses at this level had been seen in previous trials of OMV vaccines and indicates that a vaccine with several OMVs might provide partial control of disease in this age group, though the acceptability of a program with a three-dose schedule in this age group is not clear. Sequential immunization of animals with OMVs prepared from three different meningococcal strains, heterologous for PorA and PorB proteins, and the group capsular PS elicited broadly protective bactericidal antibody (59). It was proposed that this approach favored immune responses directed to the less immunodominant antigens through use of the heterologous porin immunization regime that reduced the dominance of the porin responses. Although no similar studies in humans are available, it is possible that this approach might be useful. An alternative approach to overcome the immunodominance of the porin OMPs is the overexpression of other surfaceexposed potential vaccine candidates. GNA1870 (60), also known as LP2086 (61) or factor H–binding protein (62), was overexpressed in strain H44/76 and OMVs from the mutant strain used to immunize mice. The OMV vaccine elicited broader protection against a panel of meningococcal strains than did the purified recombinant GNA1870 or conventional OMV vaccines. Upregulation of several non-dominant OMPs (including Hsf, TbpA, OMP85, and NspA) in a PorA knockout OMV vaccine, or mixing of sera raised against different OMPs, appears to have an additive effect in enhancing bactericidal responses, an approach that is now being pursued in human clinical trials (63). Since the PorA-based OMV vaccines have been strainspecific and cross-protective responses are necessary for broad control of MenB disease, preclinical research into other surface exposed membrane proteins as vaccine candidates has been

undertaken. Antigenic variability or inconsistent accessibility to antibodies across genetically different strains has proved to be problematic and few purified recombinant OMPs have so far been taken forward into clinical trials except for the transferrinbinding protein (TbpB) for which the immunogenicity was disappointing (64), and neisserial surface protein A (NspA), which was immunogenic but did not elicit functional antibodies (65). Recombinant lipoprotein 2086 was identified in a mixture of soluble OMPs following a series of fractionation, purification, and proteomics steps and its vaccine candidacy considered (61). LP2086 appears to be present among meningococcal populations in two subfamilies and combinations of LP2086 with two rPorA variants elicited bactericidal antibodies against a panel of strains. LP2086 is currently being investigated in human clinical trials as a vaccine candidate for prevention of serogroup B meningococcal disease. Although many of the OMPs that have been studied are highly variable in the structure of their surface-exposed regions, it has been observed that a relatively limited number of some proteins are expressed by the hyperinvasive clonal complexes that account for a majority of invasive disease caused by meningococci. The potential candidacy of a vaccine consisting of a relatively limited number of variants of PorA, PorB, FetA or OPa, or a combination of these proteins has been considered (39,40). Other potential candidates, discovered through genome projects are discussed below.

Lipopolysaccharide In addition to surface-expressed proteins, another major component of the cell envelope of all gram-negative bacteria, including meningococci, is LPS. The potential use of LPS as a vaccine has been impeded by concerns about toxicity, especially through lipid A (endotoxin) and the molecular mimicry of N. meningitidis glycoforms, especially lacto-N-tetraose, expressed on human cells. However, there is now compelling evidence to support the potential of meningococcal LPS as a vaccine, or a component of one. Evidence of protection by antibodies to the LPS has been documented in animals (66,67) and humans (68,69), although antibodies induced by OMV vaccines retaining LPS lacked bactericidal activity (70,71). A phase I study of detoxified LPS (immunotypes L3, L7, L9) was immunogenic, but most of the functional antibody was directed against OMPs (72). Nontoxic LPS vaccines have been also produced by conjugating the core saccharide to protein carriers, but this strategy failed to induce antibodies with bactericidal activity (73–75). A different approach, one also addressing the unproven but theoretical concern of an autoimmune response induced by lacto-N-tetraose, has resulted in vaccines based on the inner core region of LPS. This component is relatively conserved across the species, and there is strong evidence to indicate the accessibility of inner core epitopes to antibodies based on studies of in vitro grown and ex vivo organisms (76,77). A limited number of identified inner core LPS structures representative of the repertoire variants in N. meningitidis has been identified (78). These comprise phosphoethanolamine substituted at the 3, 6, or 7 position of one of two heptoses (HepII) of the inner core, or absence of phosphoethanolamine (77). Functional studies using monoclonal, polyclonal antibodies and O-deacylated immunotype L3 conjugates have shown that antibodies to inner core structures can be protective on the basis of bactericidal and passive protection assays (79,80).

Chapter 42: Meningococcal Conjugate and Protein-Based Vaccines

GENOME-BASED DISCOVERY OF NEW ANTIGENS The complete sequencing of the genomes of two meningococcal strains in the late 1990s (81,82) allowed the use of ‘‘reverse vaccinology’’ for the development of new vaccine approaches (60). Novel potential vaccine candidates were identified in silico using computer programs and subsequently expressed as recombinant fusion-proteins in Escherichia coli. The recombinant antigens were then purified and used to immunize mice. Finally, the sera obtained were tested in vitro for their ability to kill bacteria in the presence of exogenous complement. The work was performed while the sequence was in progress and by the time the sequence was published, the first screening for new antigens was also completed. Computer analysis identified approximately 600 potential antigens predicted to be surface exposed. In total, 370 of these were successfully expressed in E. coli, purified, and then used to immunize mice. Of the 370 sera tested, 29 showed bactericidal activity. Once the 29 novel antigens had been identified, they were characterized to select those that induced the best bactericidal activity against all strains. In a first approach, the genes of the novel antigens were sequenced in a panel of strains representative of the genetic variability of the worldwide population of meningococcus B. Surprisingly, many of the novel genes were found to be quite conserved among genetically diverse strains, suggesting that some of the novel proteins could be used to develop a vaccine against all serogroup B meningocci. Finally, the bactericidal activity induced by all of them was tested against a panel of genetically diverse strains. Some of the novel antigens were indeed found to induce a bactericidal activity against most of the strains tested; however, many induced bactericidal activity only against a subgroup of them. Table 3 compares the immune response against a recombinant OMP, an OMV-based

447

vaccine, and a recombinant protein found by the genomic approach. As shown, while the recombinant OMP and the OMV vaccine induce bactericidal antibodies against a subset of strains, the genome-derived antigen is able to induce immunity against all strains tested. A five-component vaccine has been constructed using promising candidates from the genome project. Antigens GNA2132 (83), GNA1870 (84), and NadA (85) were selected because they induced broadly protective bactericidal antibody and passive protection, and GNA1030 and GNA2091 because of immunity demonstrated in at least some assays (60). In this vaccine, four of the antigens were combined as two fusion proteins and mixed with NadA and adsorbed to aluminium hydroxide. In murine studies, the aluminium adsorbed 5-component vaccine induced bactericidal antibody against 75% of 85 strains selected from the major hyperinvasive meningococcal clonal complexes, ST32, ST41-44, ST8, and ST11. Higher responses and broader protection were induced when the vaccine was adjuvanted with MF59 or Freund’s complete adjuvant rather than aluminium (Fig. 1) (86). This vaccine is now being evaluated in human trials in adults and children. Preliminary data reported at the 2006 International Pathogenic Neisseria meeting in Cairns in Australia indicate that the vaccine is able to induce bactericidal antibodies in adult volunteers (Rappuoli, unpublished data). In a similar approach, Poolman and Berthet identified 30 candidate antigens from the meningococcal genome and have investigated 10 of these in combinations for their ability to induce a synergistic and protective immune response (63). Signature-tagged mutagenesis (STM) has been used to identify 73 genes, 65 of which were not known to be involved in N. meningitidis pathogenesis (87). Using a library of mutants,

Figure 1 Coverage of a panel of 85 strains representative of the global population diversity of serogroup B meningococcus by bactericidal antibodies induced by outer membrane vaccines (OMV) made using a Norwegian (Nw) or a New Zealand (Nz) vaccine, or by the recombinant 5CMCB vaccine adjuvanted with alum or other adiuvants. (A) Percentage of total strains killed by the bactericidal antibodies with a titer more than 1/128. (B) Percentage of strains killed by bactericidal antibodies with a titer more than 1/128 in each hypervirulent cluster.

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the authors of this study postulated that the mutants that failed to establish systemic meningococcal infection may have a role in the pathogenesis of N. meningitidis septicaemia. Phenotypic analysis of these genes may reveal that their products encode conserved vaccine candidate proteins, or potentially the attenuated mutants themselves could be considered as vaccine candidates (see later). DNA microarrays spanning the genome of the MC58 meningococcal strain have been used to analyze changes in gene expression during interaction with human epithelial cells (88). Host-cell contact was found to induce 347 gene alterations. One hundred and eighty-nine genes were upregulated; more than 40% of these encoded peripherally located proteins, suggesting that when in host contact, N. meningitidis undergoes substantial cell membrane remodeling. Twelve new adhesioninduced surface antigens were discovered; five were capable of inducing bactericidal activity against the homologous strain in mice. These bactericidal antigens were highly conserved across 11 isolates, including the main hyperinvasive lineages, and were able to induce some cross-protective immunity. Expression library immunization (ELI) has been used to make an expression library of the meningococcal genome for immunization of mice (89). The library was divided into 10 sublibraries (L1–L10) and used to immunize 10 groups of mice with plasmid DNA. Bactericidal antibody was induced in three groups of mice against the homologous strain, and pooled sera from one group (L8) elicited some protection from N. meningitidis challenge. After a whole-cell (live N. meningitidis) antigenic challenge, bactericidal antibodies were elicited in 9 out of 10 sublibraries (89).

COMMENSAL NEISSERIAE AND ATTENUATED BACTERIA Neisseria lactamica is a commensal species found in the human nasopharynx, predominantly in early childhood, that is thought to generate immunity that is cross-protective against meningococci. Live N. lactamica, killed whole cells, OMVs, or OMP pools when used as immunogen-protected mice against lethal challenge by a number of meningococcal serogroup B and C isolates (90,91). N. lactamica OMVs induced antibody responses that passively protected animals and are being evaluated in a phase I safety and immunogenicity study in adult volunteers (92). The protective responses appear to arise through opsonophagocytic antibody. Plasmid transformation has been used to express high levels of heterologous proteins in Neisseria flavescens (93). NspA was expressed in its native form and OMVs prepared from the modified organism induced protection in mice against a lethal challenge with N. meningitidis without bactericidal activity being present. The two-component regulatory system PhoP-PhoQ controls virulence genes of N. meningitidis. Mice infected with a live serogroup C phoP mutant of the meningococcus developed bactericidal and opsonophagocytic activity against a range of meningococci from serogroups B, Y, and W135 with different serotypes and serosubtypes (94). Further development is awaited. Two attenuated serogroup B N. meningitidis strains, YH102 and YH103, were identified by STM (see the preceding text) (95). Two mutations were inserted into each attenuated strain to reduce the possibility of reversion to wild-type B and the attenuated strains used to immunize mice. Bactericidal

antibodies developed after immunization, providing protection against homologous strains and partial protection against heterologous strains. Although the use of live attenuated bacteria provides the possibility of a more natural development of immunity if administered at mucosal surfaces, concerns about the possibility of wild-type reversion is likely to hamper development of this approach.

CONCLUSIONS PS vaccines against serogroups A, C, Y, and W135 of meningococcus have been available for decades and have been useful for immunization of at-risk groups and to control outbreaks. They have never been used for general immunization because they provide short-term immunity with no immunologic memory, and in most of the cases (with the exception of serogroup A) they do not work in infants and children. A CV against meningococcus C, licensed in the United Kingdom in 1999 has been extremely effective in controlling the disease in all ages, showing that CVs are an excellent solution for the prevention of meningococcal disease. In fact, they induce immunological memory and are efficacious at all ages. Conjugates against serogroups A, Y, W135 are now available, providing the possibility of further disease reduction in all regions with a problem from these serogroups. A CV using serogroup B meningococcal capsular PS is unlikely to be successful because of the chemical identity with human antigens, poor immunogenicity in clinical studies, even after clinical modification, and the expectations of regulatory concern over the potential for generation of autoimmunity. The use of OMVs, the use of the core structure of the LPS, or surface proteins either alone or in combination could be successful if able to induce a broadly protective bactericidal response. Several different vaccines are currently in clinical trials, providing some hope that there may be improvements in the control of meningococcal disease just around the corner.

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48.

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68. Griffiss JM, Brandt BL, Broud DD, et al. Immune response of infants and children to disseminated infections with Neisseria meningitidis. J Infect Dis 1984; 150:71–79. 69. Plested JS, Ferry BL, Coull PA, et al. Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (galE) of serogroup B meningococci measured by flow cytometry. Infect Immun 2001; 69:3203–3213. 70. Rosenqvist E, Hoiby EA, Wedege E, et al. The 5C protein of Neisseria meningitidis is highly immunogenic in humans and induces bactericidal antibodies. J Infect Dis 1993; 167:1065–1073. 71. Rosenqvist E, Hoiby EA, Wedege E, et al. Human antibody responses to meningococcal outer membrane antigens after three doses of the Norwegian group B meningococcal vaccine. Infect Immun 1995; 63:4642–4652. 72. Zollinger WD, Moran EE, Ray J, et al. Phase I safety and immunogenicity study of a meningococcal outer membrane protein, detoxified LPS vaccine. Hanasaari, Finland: Frontiers in Vaccine Research, 1991. 73. Jennings HJ, Lugowski C, Ashton FE. Conjugation of meningococcal lipopolysaccharide R type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningococcal lipopolysaccharide R type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningitidis. Infect Immun 1984; 43:407–412. 74. Verheul AF, Braat AK, Leenhouts JM, et al. Preparation, characterisation, and immunogenicity of meningococcal immunotype L2 and L3,7,9 phosphoethanolamine group-containing oligosaccharide-protein conjugates. Infect Immun 1991; 59:843–851. 75. Verheul AF, Van Gaans JA, Wiertz EJ, et al. Meningococcal lipopolysaccharide (LPS)-derived oligosaccharide-protein conjugates evoke outer membrane protein- but not LPS-specific bactericidal antibodies in mice: influence of adjuvants. Infect Immun 1993; 61:187–196. 76. Andersen SR, Guthrie T, Guile GR, et al. Cross-reactive polyclonal antibodies to the Inner Core of lipopolysaccharide from Neisseria meningitidis. Infect Immun 2002; 70:1293–1300. 77. Plested JS, Makepeace K, Jennings MP, et al. Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis. Infect Immun 1999; 67:5417–5426. 78. Gidney MA, Plested JS, Lacelle S, et al. Development, characterization, and functional activity of a panel of specific monoclonal antibodies to inner core lipopolysaccharide epitopes in Neisseria meningitidis. Infect Immun 2004; 72(1):559–569. 79. Plested JS, Harris SL, Wright JC, et al. Highly conserved Neisseria meningitidis inner-core lipopolysaccharide epitope confers protection against experimental meningococcal bacteremia. J Infect Dis 2003; 187(8):1223–1234. 80. Pavliak V, Fortuna-Nevin M, Monteiro M, et al. Neisseria meningitidis LOS conjugate vaccine against meningococcal disease. 14th International Pathogenic Neisseria Conference, Milwaukee, September 5–15, 2004. 81. Tettelin H, Saunders NJ, Heidelberg J, et al. Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000; 287:1809–1815. 82. Parkhill J, Achtman M, James KD, et al. Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 2000; 404:451–452. 83. Welsch JA, Moe GR, Rossi R, et al. Antibody to genome-derived neisserial antigen 2132, a Neisseria meningitidis candidate vaccine, confers protection against bacteremia in the absence of complement-mediated bactericidal activity. J Infect Dis 2003; 188(11): 1730–1740. 84. Masignani V, Comanducci M, Giuliani MM, et al. Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J Exp Med 2003; 197(6):789–799. 85. Comanducci M, Bambini S, Brunelli B, et al. NadA, a novel vaccine candidate of Neisseria meningitidis. J Exp Med 2002; 195(11):1445–1454.

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43 Post-Licensure Impact of Haemophilus influenzae Type b and Serogroup C Neisseria meningitidis Conjugate Vaccines in Industrialized Countries Elizabeth Miller, Helen Campbell, and Mary Ramsay Immunisation Department, Centre for Infections, Health Protection Agency, London, U.K.

INTRODUCTION Polysaccharide-protein conjugate vaccines are established tools for the prevention of serious disease caused by encapsulated bacteria. Conjugate vaccines to prevent disease caused by Haemophilus influenzae type b (Hib) and serogroup C meningococcus (MCC) have been used in routine national immunization programs over a number of years. In this chapter, we review development and implementation of these vaccines, and present data from industrialized countries demonstrating their striking impact and the continued need for effective enhanced surveillance post-vaccine introduction.

THE BACTERIA H. influenzae and Neisseria meningitidis are unrelated bacteria, which share a number of characteristics, the most obvious being their ability to cause bacterial meningitis and septicemia in humans. H. influenzae is a gram-negative coccobacillus, which colonizes the human oropharynx. A minority of these organisms have polysaccharide capsules. The encapsulated strains are characterized on the basis of the antigenic properties of the capsule. Of the six serologically identified types (designated a–f), organisms with the type b capsule (Hib) are the most virulent. Since humans are the only biologically relevant reservoir of H. influenzae, transmission of the organism occurs by person-toperson spread, usually through direct or indirect exchange of oropharyngeal secretions. After introduction into the oropharynx, H. influenzae may establish relatively long-term colonization (often lasting several months or more) or, primarily in the case of Hib, may invade through the mucosa and enter the bloodstream. Once the organism gains access to the bloodstream, it multiplies and may seed other sites, such as the meninges, joint spaces, or soft tissue. If Hib remains unchecked, clinical sepsis may follow, with or without manifestations of localized disease. Hib also causes lower respiratory tract infection, presumably by aspiration or direct extension from the oropharynx; only a minority of these lower respiratory tract infections result in bacteremia. N. meningitidis is a gram-negative coccus with a polysaccharide capsule. The capsular polysaccharides of meningococcus define the serogroup. Thirteen serogroups have been identified, of which six (A, B, C, W135, X, and Y) commonly cause invasive human disease (1). Serogroups A, B, and C are the most important in terms of morbidity and mortality world-

wide, accounting for 90% or more cases of clinical disease. All three of these serogroups may cause endemic disease. Serogroup A strains have caused most of the major outbreaks, especially in the African ‘‘meningitis belt.’’ Serogroups B and C have been associated with outbreaks in the developed world, usually with substantially lower incidence rates than those in the meningitis belt. Humans are the only recognized host for N. meningitides, and there is no known animal or environmental reservoir. N. meningitidis, similar to Hib, colonizes the human oropharynx. Meningococcus is highly adapted to this commensal existence in humans, with a range of strategies for evasion of the immune response. The carrier state may last for a few days to months; it provides a reservoir for infection and enhances the immunity of the host. As with Hib, invasive disease—primarily meningitis and/or sepsis—may follow carriage, but meningococcus is a less common cause of pneumonia.

EPIDEMIOLOGY OF DISEASE: HIB In the 1940s, Hattie Alexander, a pediatrician and microbiologist working in New York City, demonstrated that if appropriate bacteriological methods were used, the fastidious bacterium Hib was commonly isolated from the cerebrospinal fluid (CSF) and blood of infants with purulent meningitis (2,3). With more widespread availability of appropriate bacteriological methods and the culturing of blood and ordinarily sterile body fluids (e.g., CSF, synovial fluid, etc.) from infants with focal infections and suspected sepsis as a standard of care, in the 1960s and 1970s, Hib came to be recognized as a major cause of severe invasive pediatric infections in industrialized countries in North America (4–6), northern and western Europe (7–13) and Australia (14), and New Zealand (15). Indeed, prior to the introduction of routine immunization of infants and toddlers with Hib conjugates in these industrialized countries, Hib was the most predominant agent responsible for bacterial meningitis and a major cause of various other invasive infections (e.g., septic arthritis, pericarditis, periorbital cellulitis), as well as pneumonia with empyema and epiglottitis among children less than five years of age. In the United States, for example, in 1987, prior to infant Hib conjugate vaccine introduction, the incidence of invasive Hib disease was 41 cases per 100,000 children <5 years of age, with a peak incidence from 6 to 11 months of age. In the late 1970s and early 1980s, approximately 20,000 invasive Hib cases occurred annually prior to the

Chapter 43: Post-Licensure Impact of Hib and Neisseria meningitidis CVs

453

Figure 1 The distribution of meningococcal serogroup C cases and deaths by age, England only, 1998/1999. Source: Courtesy of Health Protection Agency.

Hib conjugate vaccine era, including *12,000 cases of meningitis, and approximately 6% to 10% of cases were fatal. The cumulative risk was such that 1 in 200 U.S. children developed an invasive Hib infection prior to reaching age five years. Incidence rates among children <5 years of age were somewhat lower in some European countries, and in Scandinavia the peak incidence occurred among toddlers rather than infants. In the pre-Hib conjugate vaccine era, certain subpopulations, such as Navajo and Apache native Americans (16,17) and Alaskan Inuits (18,19) in the United States and aboriginals in Australia (20–23), experienced incidence rates of invasive Hib disease, including meningitis, that were ~3- to 6-fold higher than children in the general population. The peak incidence in these subpopulations tended to occur somewhat earlier than in the general population (24). The introduction of routine Hib immunization in industrialized countries led to a striking decline in the incidence of invasive disease in these high-risk subpopulations (25–27), as well as in the general pediatric population (28–30).

EPIDEMIOLOGY OF DISEASE: SEROGROUP C MENINGOCOCCUS In contrast to Hib, which causes a relatively stable burden of childhood meningitis in a given country, meningococci not only contribute a continuing burden of endemic disease but also cause epidemics with widely varying rates, occurring at unpredictable intervals. Worldwide, there are around 1.2 million cases of endemic and epidemic meningococcal disease each year, with an estimated 135,000 deaths (31). The disease can occur anywhere, but the largest and most frequent epidemics arise in the African meningitis belt, where epidemic waves of meningococcal disease occur every 5 to 12 years, usually because of serogroup A organisms, with serogroup C and W135 strains playing a smaller role. Certain factors are thought to increase susceptibility to meningococcal infection including climate, crowded living conditions, upper respiratory tract infection, and waning population immunity. There is clear seasonal variation, with the highest incidence of endemic and outbreak disease in the winter months. Disease onset is often sudden, and even with correct treatment, individuals may be left with severe disabling

sequelae, in particular brain damage and loss of limbs. The case fatality rate is high—10% to 20% of all cases of meningococcal disease die—but varies with serogroup, clinical presentation (meningitis, sepsis), and the availability of prompt antibiotic treatment. In developed countries, serogroups B and C predominated as a cause of invasive disease prior to the availability of a vaccine against meningococcal C infection. From the mid-1980s and early 1990s, a disproportionate increase in cases of meningococcal disease caused by serogroup C was observed in a number of European countries including England and Wales, Greece (32), Spain (33), and the Republic of Ireland (34). In Canada (35) the proportion of serogroup C cases increased significantly from 24% in 1985 to 65% in 1992 against a background of increasing incidence of all meningococcal infection. The United States had a relatively low incidence of meningococcal disease, but outbreaks of serogroup C meningococcal disease also began to be observed more frequently from the early 1990s (36). In the absence of MCC immunization, the peak incidence of meningococcal C disease throughout the developed world is in children aged under two years of age, with a secondary peak in individuals aged between 15 and 18 years (Fig. 1). The age distribution has changed during some epidemics with an increase in the proportion of cases observed in teenagers and young adults. This is important as, unlike disease due to meningococcal B or Hib infection, the death rates are highest in those aged between 15 and 18 years (Fig. 1). The case fatality rate for serogroup C disease is generally higher than that for serogroup B (37).

HIB VACCINE DEVELOPMENT Immunology and Development The key to Hib vaccine development was the recognition that serum IgG antibodies to polyribosylribitol phosphate (PRP), the capsular polysaccharide that covers Hib bacterial cells, is associated with complement-mediated bactericidal activity and with protection against invasive disease. The challenge to be met then was to discover how to actively elicit protective levels of IgG anti-PRP in young infants. An early-generation vaccine strategy, based on immunizing toddlers and preschool

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In the United States, primary immunization of infants with PRP-T is recommended as a three-dose regimen, with doses given at two, four, and six months of age, while immunization with PRP-OMP is administered as a two-dose regimen at two and four months of age. A booster dose is given to toddlers at 12 to 15 months of age.

children with purified unconjugated PRP, provided an intermediate step on the way. Infants and toddlers <18 months of age do not mount responses to unconjugated PRP, since, like other polysaccharides, it functions as a T-independent antigen. However, preschool children above 24 months of age do manifest responses to unconjugated PRP. In a large-scale controlled field trial (39) in Finland in which infants and children 3 months to 5 years of age were randomly allocated to receive unconjugated PRP or unconjugated meningococcal polysaccharide, the PRP vaccine did not protect infants and toddlers who were less than 18 months of age at the time of vaccination. By contrast, in the Finnish field trial, PRP conferred 90% efficacy in preventing bacteremic Hib disease among children who were 18 to 71 months of age at the time of vaccination. On the basis of the Finnish field trial data and on evidence of safety and immunogenicity in toddlers in the United States, the unconjugated vaccine was licensed by the FDA in 1985 and was recommended for routine immunization of children at 24 months of age. It was also recommended for administration to toddlers at 18 months of age if they were attending day care or were otherwise considered at increased risk of developing invasive Hib disease. However, post-licensure surveillance indicated that the impact of the unconjugated PRP vaccine was generally modest, and five different case control studies showed widely divergent results, with one study showing no efficacy and even suggesting an increased risk post vaccination (38–42). Conjugate vaccines consisting of PRP covalently linked to a carrier protein fundamentally alter how the immune system interacts with the polysaccharide. When conjugated, PRP functions as a T-dependent antigen capable of eliciting serum antiPRP antibodies in young infants and conferring immunological memory (that allows boosting) when reinforcing doses are administered (42–44). PRP conjugated to diphtheria toxoid (PRP-D) was the first conjugate vaccine to be licensed in the United States, in 1987 and was recommended for use in toddlers >15 months of age. In large-scale, randomized, controlled field trials, this Hib conjugate was efficacious in a study in Finland (45) but did not protect Inuit native Americans in a trial in Alaska (46). The first conjugates for use in young infants >2 months of age in the United States were licensed in 1988 and 1989, including Hib oligosaccharide conjugated to CRM197 (mutant diphtheria toxin) and PRP conjugated to the outer membrane protein of group B meningococcus (PRP-OMP), respectively. The efficacy of these two vaccines in preventing invasive Hib disease in U.S. infants was demonstrated in largescale controlled field trials (47,48). A third vaccine for use in young infants, PRP conjugated to tetanus toxoid, was licensed in 1993 on the basis of the ability to generate equivalent serum antiPRP responses in infants, compared with the other two conjugate vaccines licensed for infants (49,50).

IMPACT OF HIB IMMUNIZATION REGIMENS United States Following the introduction of routine immunization with the Hib conjugate vaccines in the late 1980s, the incidence of invasive Hib disease plummeted by >90% within just three years (29). By 1999, the reduction was 99%, compared with the 1987 baseline incidence rates (23). Marked reductions in incidence of invasive Hib disease were noted in high-risk native American populations (25–27), as well as in the general population (28–30). However, as vaccination with Hib conjugate became widespread, some subgroups within the general population came to be identified that had low immunization rates (51). Moreover, the achievement and maintenance of control of Hib disease among Alaskan natives proved to be particularly challenging (25,52).

The Hib Experience in England and Wales The United Kingdom introduced Hib conjugate vaccine into its routine childhood program in October 1992 to be given at two, three, and four months of age (53). A catch-up program was undertaken at the same time for other children under four years of age with Hib vaccine given as a single dose in those over one year. Unlike some other countries where Hib vaccine had been introduced, a booster dose was not recommended after the primary course. Hib vaccine coverage quickly reached high levels and within a year had reached 92% for the primary schedule (54). Uptake for children aged one to four years was around 75% (55). The number of confirmed cases of invasive Hib disease in children under five years of age in England and Wales fell from 744 in 1991/1992, before Hib vaccine was introduced, to 37 by 1994/1995 (Table 1); a reduction of 95%. Follow-up studies at that time showed that anti-Hib antibodies remained above protective levels in most children for up to 12 months after vaccination (56) but were subsequently shown to fall significantly after this period (57). The high vaccine effectiveness calculated in the latter study was misleading as it was based on measures of both direct and indirect protection. The catch-up campaign played a significant part in the success of the introduction of Hib vaccine (58). Targeting the one- to four-year age group resulted in high and prolonged

Table 1 Confirmed Cases of Invasive Hib Disease in England and Wales, 1991/1992 to 2007/2008 Age group

1991/ 1992/ 1993/ 1994/ 1995/ 1996/ 1997/ 1998/ 1999/ 2000/ 2001/ 2002/ 2003/ 2004/ 2005/ 2006/ 2007/ 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

<1 yr 1–4 yr 5–9 yr 10–14 yr 15þ yr Not known

311 433 16 15 66 5

193 408 11 7 80 16

25 33 12 1 43 4

15 22 7 1 30 2

14 17 3 1 31

Total

846

715

118

77

66

Source: Courtesy of Health Protection Agency.

13 19 5 21 3 61

9 13 1 2 18

9 17 0 20

12 34 4 2 35

15 52 10 2 34

22 88 10 3 61 2

30 131 25 4 109 2

22 40 22 4 108 2

13 21 8 5 82 2

8 30 1 5 64

13 33 3 4 54 1

11 18 2 1 52

43

46

87

113

186

301

198

131

108

108

84

Chapter 43: Post-Licensure Impact of Hib and Neisseria meningitidis CVs

direct protection in this cohort; probably due to the high antibody levels achieved when children aged one year or older are vaccinated, even with a single dose (58). Toddlers have the highest rates of asymptomatic pharyngeal carriage of Hib (59,60), and Hib conjugate vaccine has been shown to dramatically reduce Hib carriage (61–63). Thus, the U.K. program, including the catch-up campaign, also provided high levels of indirect protection (herd immunity) by reducing Hib transmission across all age groups (66). From 1999, the number of reported invasive Hib disease cases began to rise in children under five years (65). Increases in older age groups became apparent from 2001. A major factor was the greater than expected decline in vaccine effectiveness among children vaccinated in infancy (67) and most cases arose in fully immunized children (65). In children immunized in infancy between 1992 and 2002, vaccine effectiveness was estimated to decline from 61% in the first two years after vaccination to 27% after that (p ¼ 0.022) (67). In 2000 and 2001, there were supply problems with diphtheria-tetanuspertussis-Hib (DTP-Hib) combination vaccines containing whole-cell pertussis (wP). This led to the use of combined vaccines containing acellular pertussis (aP) vaccine (68), of which several have been shown to have reduced Hib immunogenicity (69,70), particularly at an accelerated immunization schedule such as that used in the United Kingdom. The reduced immunogenicity of these vaccines was not considered to be clinically relevant, as immune memory was still generated (71,72). A U.K. case control study, however, showed that children fully vaccinated with DTaP-Hib vaccine had a higher risk of vaccine failure than those fully vaccinated with a DTwPHib preparation, with increasing odds of disease for each dose of the DTaP-Hib vaccine (73). This was consistent with the reduced vaccine effectiveness in children immunized when the DTaP-Hib vaccine was in use (67). The decline in vaccine effectiveness after infant vaccination was still apparent, however, in infants who had only received DTwP-Hib vaccine (74). The number of cases of invasive Hib disease in adults also rose from 20 in 1998/1999 to 109 in 2002/2003 (Table 1), higher than the average of 75 cases per year in the pre-vaccine era. This was attributed to a reduction in opportunities for natural boosting of immunity after routine infant immunization against Hib was introduced in 1992, resulting in a significant decline in serum antibody levels against Hib in adults (66). The resurgence of invasive Hib disease in younger children from 1999 then increased the risk of exposure to the organism and, therefore, the risk of infection among susceptible adults (66). These observed increases in Hib disease culminated in a decision to conduct a Hib booster campaign from May 2003. All children between six months and four years of age were offered one additional dose of Hib vaccine. National vaccine coverage for the 6- to 12-month age group was 72% and 63% in those aged 13 to 48 months (68). The 2003 booster campaign had a major and rapid impact on disease in the targeted age group. This control was achieved by targeting the age group with the largest increase in cases, many of whom had received the less effective DTaP-Hib vaccine in their primary schedule. It was also suggested that this age group had become recolonized with Hib prior to the booster campaign (64), therefore becoming a source of transmission to others. This was supported by a carriage rate of 2.1% observed in one study of preschool children in 2003 (64), higher than that seen at other points during the post-vaccine era (63).

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The decline in Hib cases in vaccinated age groups was soon followed by a reduction in older children and adults, suggesting that the booster campaign had again contributed significantly to indirect protection in the non-vaccinated. In 2003, the implicated DTaP vaccine was withdrawn and the use of DTwP vaccines was resumed. In September 2004, the recommended vaccine for infants was changed to a vaccine containing DTaP, inactivated polio, and Hib. This combination vaccine had a different aP component from the one implicated in the increase and had been shown to have a satisfactory immune response to the Hib component (75). In September 2006, a booster dose of the Hib conjugate vaccine was introduced at 12 months of age into the national schedule. Following the introduction of the routine booster dose, the incidence of invasive Hib disease has fallen again in toddlers and, presumably by reducing asymptomatic carriage, has led to a slow decline in older children and adults (68).

The Hib Situation in Other European Countries The pre-vaccine incidence of Hib meningitis varied widely in Europe from 8 to 27 cases per 100,000 children under five years of age, with incidence of all invasive Hib diseases varying from 12 to 52 cases per 100,000 children under five years of age. Within the European Union countries, pre-vaccine rates were highest in Scandinavia and Iceland, with low rates in Mediterranean countries and differences within countries were also often observed (76). The first country to introduce national vaccination against Hib was Finland in 1986, followed by Iceland in 1989. Most other western European countries introduced vaccination between 1990 and 1996. The schedule used for Hib often followed the schedule used for DTP vaccination— with most countries having three doses in infancy followed by a booster in the second year of life. Exceptions to this included many Scandinavian countries and Italy, where two doses in infancy are followed with a booster at around 12 months of age, and the United Kingdom and the Republic of Ireland where only three doses in infancy were used initially (77). Despite the use of different vaccines and the achievement of different levels of coverage, all countries have seen a major reduction in the annual attack rate of Hib within a few years of vaccine introduction (78–88). In most European countries, the incidence of Hib disease has remained stable and low in the post-vaccine era (77). In addition to the United Kingdom, however, two other countries have experienced sustained increases in Hib incidence after a long period of good control. In Ireland, cases (incidence) of Hib disease fell from approximately 100 per year in the late 1980s (2.84/100,000 total population) to 10 cases or less per year by 2002 (0.26/100,000 total population). From 2003, however, the overall number of Hib cases and those in children under 15 years of age started to increase slightly. Cases in fully vaccinated children increased from a baseline of between 1 and 4 cases annually to 10 cases being reported between January and September 2005 (89). This increase in vaccine failures in Ireland was probably due to similar causes as that in the United Kingdom. Infants in Ireland were routinely immunized at two, four, and six months (with no booster), and DTaP-Hib combinations were introduced in 2001. In response to the increase, a Hib booster campaign commenced in Ireland in November 2005. The other country that has experienced an increase is the Netherlands. Unlike the United Kingdom and Ireland, the

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Netherlands had always given a booster dose of vaccine, and DTaP-Hib combinations were not introduced until 2005. The number of invasive Hib isolates referred to the national reference laboratory increased from the lowest level in 1999 (15) to 49 by 2004. The number of vaccine failures also increased from an annual number below 5 to between 10 and 15 per year after 2002. It has been suggested that this increase was probably due to waning protection, even in the presence of a booster, possibly exacerbated by reduced natural boosting from lower rates of asymptomatic carriage (90).

MENINGOCOCCAL C VACCINE DEVELOPMENT Immunology and Development The pathophysiology and epidemiology of serogroup C meningococcal disease are similar in some respects to that of Hib and present some of the same difficulties for vaccine development. Capsular antigens have been crucial in the development of licensed vaccines against meningococcal C disease (91), and highly purified, high–molecular weight meningococcal capsular polysaccharides were shown to be safe and highly immunogenic in adults and older children in the 1960s. These are the basis of the bivalent A and C and tetravalent A, C, W135, and Y meningococcal polysaccharide vaccines. The highest attack rate for serogroup C meningococcal disease in the developed world occurs in infancy, but, as for Hib, meningococcal capsular polysaccharides are poorly immunogenic in young children. This is because meningococcal capsular polysaccharides usually act as T cell–independent antigens. T-independent responses are age dependent, not generally occurring before 18 months of age, and do not induce immunological memory (92). Consequently, licensed polysaccharide vaccines are ineffective in protecting young children against group C disease and do not provide long-term protection. The relatively recent development of Hib conjugate vaccine was therefore closely followed by the development of meningococcal conjugate vaccines. The vaccines are made from oligosaccharides derived from purified capsular polysaccharides that are chemically conjugated, using different methods, to tetanus toxoid or diphtheria CRM197 carrier proteins to convert them into T-dependent antigens (93).

Correlates of Protection in Meningococcal C Conjugate Vaccines Serum bactericidal activity (SBA) was established as the natural correlate of protection for meningococcal disease in the 1960s (94,95). Trials with serogroup C plain polysaccharide vaccine confirmed that SBA was also a correlate of vaccine-induced protection, with poor SBA responses in children less than 24 months and increasing SBA response with age (96). A broad correlation between immunogenicity and effectiveness of the group C polysaccharide vaccine with age was shown in separate trials (97–99). These studies established that when SBA activity was present, an individual was protected. Since SBA levels wane after vaccination, it was also known that protection was short lived. Ability to induce SBA subsequently became a WHOrecommended correlate of protection (100), and so serogroup C polysaccharide vaccines were licensed without an efficacy trial. Vaccine-induced SBA was accepted as a correlate of protection for individuals over 24 months, but MCC vaccines were designed to protect children under two years of age. It, therefore, seemed logical that, if MCC vaccines elicited a SBA response in infants, like plain polysaccharide vaccines did in older age

groups, they would protect while SBA levels remained. This assumption was the basis of licensure of MCC vaccines for infants and toddlers in the United Kingdom (101) and was validated by later post-marketing effectiveness data. Clinical trials of MCC vaccines found them to be immunogenic in all age groups with reactogenicity profiles in line with other routine vaccines and no serious adverse events identified (102–106). MCC vaccines are T cell–dependent antigens shown to induce a booster response up to four years after completion of infant immunization via immune memory (107). It was postulated that they would provide long-term protection as a result of rapid boosting of SBA levels on exposure. This assumption was based on the U.K. experience with Hib conjugate vaccines, which, until the resurgence in 2000, were thought to be providing long-term protection via immune memory despite waning Hib antibody levels (108). It was subsequently realized that the control of Hib disease in the United Kingdom was due to a reduction of carriage and induction of herd immunity and that the direct protection from Hib conjugate vaccine given in the first year of life was short lived (68,109). Had it been known at the time of licensure of MCC vaccines that immune memory induced by Hib conjugate vaccines given in infancy was not generating long-term protection, the assumption that MCC vaccines would protect in the long term despite loss of SBA would not have been made.

IMPACT OF MCC VACCINES IN ENGLAND AND WALES Introduction of MCC Vaccine in England and Wales As with many other developed countries, the incidence of all meningococcal infections increased through the 1990s in England and Wales. The incidence rose from 2.8 per 100,000 in the 1990/1991 epidemiological year (running from July to June) to 5.3 per 100,000 in 1998/1999. The rise was in part due to better ascertainment following the wide availability of more sensitive polymerase chain reaction (PCR) methods (110,111), however, there was a true rise in the level of endemic serogroup C infection, which increased proportionately more than other serogroups. In November 1999, the United Kingdom became the first country in the world to introduce MCC immunization into the routine infant schedule. A phased national MCC immunization catch-up campaign also began in November 1999, targeting all children under 18 years of age (12 million in England and Wales). Recorded MCC vaccine coverage for school children aged 5 to 17 years was 85%, and coverage for children aged 5 months to 4 years of age was 78%. Routine infant immunization rapidly reached levels comparable to other vaccines given in the primary schedule and was 93% in July 2006 (112).

Impact on Disease in England and Wales Cases of meningococcal B and C infections in all age groups are shown in Figure 2 for England and Wales from the 1998/1999 epidemiological year to 2007/2008. This graph clearly demonstrates that cases of Group B disease continued to occur at levels previously recorded, with natural variation by year, while the level of group C disease was markedly reduced within a year of the MCC campaign. Group C cases decreased by 97% overall between 1998/1999 (955 cases), before MCC vaccine was introduced, and 2007/2008 (29 cases). Deaths also fell strikingly in this period by 99%, from 118 serogroup C deaths in 1998/1999 to only 1 recorded death in 2007/2008.

Chapter 43: Post-Licensure Impact of Hib and Neisseria meningitidis CVs

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Figure 2 Total cases of laboratory-confirmed meningococcal B and C disease in England and Wales between 1998/1999 and 2007/2008. Source: Courtesy of Health Protection Agency.

Table 2 Age Distribution of Meningococcal Serogroup C Cases in England and Wales, 1989/1999 to 2007/2008 Age

1998/ 1999

1999/ 2000

2000/ 2001

2001/ 2002

<1 yr 1 yr 2 yr 3–4 yr 5–8 yr 9–10 yr 11–14 yr 15–19 yr 20–24 yr 25þ yr Not known

105 80 47 84 75 20 83 207 44 194 16

66 56 50 124 100 23 66 119 52 235 1

20 11 19 15 27 1 10 38 66 205

6 9 7 7 5 1 5 17 31 122 1

Total

955

892

412

211

2002/ 2003

2003/ 2004

2004/ 2005

2005/ 2006

2006/ 2007

2007/ 2008

2 2

1 1 1 2 1 1

1

2 1 1

1

6 5 3 5 5

9 1

9 17 71

1 1 7 41

3 3 29

1 2 3 19

121

64

42

30

3 1

2

2 1

1 2 19 1

4 2 19

29

29

Source: Courtesy of Health Protection Agency.

This impact has been apparent in all age groups (Table 2), including those who have not been targeted for immunization with MCC vaccine, the over 25s and those under 3 months of age who are not old enough to have completed the primary course (113). The fall in these groups has occurred as a result of reduction in carriage and a herd immunity effect, which are now known to be key to the vaccine impact (114).

MCC Vaccine Effectiveness Estimates of vaccine effectiveness in the first year were high in all age groups (115). Subsequent analyses up to six years after vaccination showed a marked decline in those vaccinated in infancy under the routine schedule, while high effectiveness was maintained in older age groups (113,116). This waning of vaccine effectiveness in routinely vaccinated infants was considered to be of concern, but the actual number of cases of meningococcal C disease in these cohorts remained low

because of high levels of indirect protection (117). On the basis of modeling studies (118) and the U.K. experience with the Hib catch-up program, it was felt that this herd immunity effect was likely to persist for several years. Studies in infants and adolescents showed that the decline in effectiveness in infants paralleled the decline in SBA, while in adolescents, SBA titers showed little evidence of a decline with time, mirroring the sustained high effectiveness in this age group (120,121). The failure of MCC vaccines to protect after SBA titers decline suggests that the ability to mount a booster response via induction of immune memory does not always result in long-term protection, as was also shown for Hib conjugate vaccines. As a result, the MCC primary vaccine schedule was changed to two doses at three and four months of age in England and Wales in September 2006, with booster doses recommended for both MCC and Hib conjugate vaccines in the first year of life, after which antibodies are expected to persist longer.

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Table 3 Routine Use of MCC Vaccine in European Union Countries As at October 2007

Country

Year of introduction to routine schedule

Recommended routine schedule

Catch-up cohort

Belgium Germany

2002 2006

12 mo 12–23 mo

Iceland Ireland

2002 2000

Netherlands Portugal Spain United Kingdom

2002 2006 2000 1999

6 and 8 mo 2, 4 and 6 mo 12–15 mo 14 mo 3, 5, and 15 mo 2, 4, and 6 mo 2, 3, and 4 mo 3, 4, and 12 mo from September 2006

1–17 yr age groupa No formal catch-up but included in general recommendations for older children 6 mo to 19 yr Under 23 yr 1–18 yr age group Under 10 yr in 2006; 10–18 yr in 2007 7 mo to 19 yrb Under 18 yr in 1999/2000 19–25 yr in 2001/2002

a

The Wallonie region targeted children aged one to six years only in their catch-up. In 15 of 19 Spanish regions. Source: Courtesy of European Union Invasive Bacterial Infections Surveillance Network. b

Meningococcal Isolates and Capsule Switching Concerns were raised about the possibility of capsule switching arising from selection pressure by MCC vaccine on serogroup C strains (119). All invasive strains of N. meningitidis received at the HPA Meningococcal Reference Unit (MRU) are typed using monoclonal antibodies to determine their serotype and serosubtype. These data have been used to monitor whether there is any evidence of serogroup C characteristics appearing in isolates belonging to other serogroups. There is no evidence of overall increases in group B organisms with serosubtypes corresponding to the more common C strains and, therefore, no evidence of capsule switching occurring to date (122).

IMPACT OF MCC VACCINE IN OTHER EUROPEAN COUNTRIES Other European countries introduced routine MCC vaccination from 2000 onward; by October 2007, this included Belgium, Germany, Iceland, Ireland, the Netherlands, Portugal, and Spain, with schedules as detailed in Table 3. The vaccine has also been introduced on a voluntary or selective basis from 2001 in the Czech Republic, Greece, Hungary, Italy, Norway, Poland, Sweden, and Switzerland (123). The impact in countries using MCC vaccine is summarized in Figure 3. This illustrates the marked reduction in disease in European countries using MCC vaccine, with a consistent pattern of disease in countries not using the vaccine. There was a decrease in cases of serogroups B and C between 1999 and 2004 in countries using MCC vaccine, however, group C disease decreased by over 80%, while serogroup B fell by less than 30% (123). Studies from Spain, where there is a three-dose schedule, completed before one year of age, substantiated the findings from England and Wales that protection falls over time after a primary MCC immunization schedule under one year of age (124,125). Cases in the Netherlands fell from 276 in 2001 to 17 in 2004 (a reduction of 94%) (126). There have been no reported vaccine failures in the Netherlands up to February 2007 (127), and this supports the data from England and Wales indicating that a single dose in the second year of life affords longer protection. However, this observation could also be explained by high levels of herd immunity conferred by the large catchup campaign. This herd immunity also explains the reduction

Figure 3 Incidence of serogroup C invasive meningococcal disease by routine MCC vaccine use and epidemiological year in reporting European countries. Source: Courtesy of European Union Invasive Bacterial Infections Surveillance Network.

in cases amongst those under 14 months who had not been targeted with MCC vaccine in the Netherlands. MCC vaccine was launched in the Republic of Ireland at the beginning of October 2000 for everyone under the age of 23 years (34). The incidence of group C disease fell dramatically from 132 cases (3.6 per 100,000) in 1999 (128) to 4 cases in 2006 (0.1 per 100,000), a 97% reduction (129).

IMPACT OF MCC VACCINE IN NON-EUROPEAN INDUSTRIALIZED COUNTRIES Canada In Canada, an increase in serogroup C meningococcal disease in the early 1990s led to mass campaigns with serogroup A and C polysaccharide vaccine in many areas (130,131). Disease rates fell in those targeted for vaccination but remained high in older age groups. In 2001, evidence of waning protection (132,133) was followed by a further increase in disease rates. Therefore, a mass campaign with MCC vaccine was undertaken to control the epidemic, and high levels of protection were demonstrated

Chapter 43: Post-Licensure Impact of Hib and Neisseria meningitidis CVs

(134). Since 2001, the Canadian National Advisory Committee on Immunizations (NACI) has recommended MCC vaccine for all children under five years of age, adolescents, and young adults (135). The NACI recommendations have not been implemented consistently throughout the country. By July 2005, 12 of 13 provinces had implemented a universal MCC vaccination program at various ages. Initial surveillance data indicated that MCC vaccine may be having an impact on the epidemiology of serogroup C disease in Canada, but surveillance is continuing (136).

Australia In 2002, the proportion of meningococcal disease due to serogroup C infection increased to around 41% in Australia (137). Serogroup C disease was observed to be more common in adolescents and young adults and was associated with a high case fatality rate. MCC vaccine was introduced early in 2003 and was administered as a single dose at 12 months of age as part of the routine schedule. It was also offered as part of a catch-up campaign to all those under 20 years of age (138). The routine schedule was set at 12 months because the burden of group C disease was predominantly in older children, teenagers, and adults. The number of laboratory-confirmed cases of meningococcal C disease in Australia fell from 213 in 2002 to 50 in 2005; a reduction of more than 75%.

United States In the United States, the main meningococcal serogroups up to 2007 were B, C, and Y, with each being responsible for approximately one-third of all cases; the proportion of cases caused by each serogroup varied by age. A public health strategy ideally needed to target the relatively high level of serogroup Y disease and, in January 2005, a tetravalent meningococcal polysaccharide-protein conjugate vaccine (MCV4) was licensed. The vaccine contained capsular polysaccharide from serogroups A, C, Y, and W-135 and was licensed for use in individuals aged between 11 and 55 years. Analysis in the United States suggested that routine immunization with MCV4 in adolescents would be more cost effective than vaccination of toddlers or infants (139). This was due to the higher disease rates and the high level of carriage in adolescents. In 2005, routine vaccination of young adolescents (aged 11 to 12 years) was recommended with MCV4 at the preadolescent health care visit (140). For adolescents who had not previously received MCV4, vaccination before high school entry (at approximately age 15 years) was recommended as an effective strategy to reduce meningococcal disease incidence among adolescents and young adults.

CONCLUSION The success of Hib conjugate vaccines in countries that have immunized the majority of children has been marked, ranging from elimination of disease in nations with very high immunization coverage to declines of over 95% in countries with somewhat lower immunization rates. Similarly, data on impact of the MCC vaccine are impressive. Worldwide use of Hib and MCC vaccines could lead to the virtual elimination of these infections. However, major obstacles stand in the way of achievement of this goal. Conjugate vaccines are currently expensive, and implementation of standard regimens into many national immunization program schedules poses a

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daunting challenge for available health care resources, particularly in developing countries. Progress is needed in reducing production and distribution costs. Equally important is interaction between ministries of health and immunization partners to enhance support for conjugate vaccine immunization programs. Accurate estimates of the burden of disease as well as clearly defined examples of impact are needed to contribute to the political will to use these vaccines. The experience with minor resurgences of Hib disease, however, in both developed (Alaska, United Kingdom, Ireland, the Netherlands) and developing countries [Gambia (141)] and the waning MCC vaccine effectiveness after a primary course in infancy emphasize the importance of continued surveillance for disease in countries that have introduced these vaccines. Such surveillance is essential for further understanding of the impact of vaccination as observed with recognition of the importance of herd immunity and reduction of carriage in the population impact of Hib and MCC vaccines. It is also crucial to enable assessment of the need for different strategies, for example, use of a booster dose of Hib and MCC vaccines. Finally, efforts to enhance routine immunization coverage, including improved logistics and decreasing dropout rates of children returning for multidose regimens, are critical. Most of these challenges are common to all childhood immunization programs, however, and the existence of these effective tools for elimination of a much-feared disease of childhood can contribute to the will to overcome these obstacles for all vaccines.

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44 Haemophilus influenzae Type b Disease Burden and the Impact of Programmatic Infant Immunization in Developing Countries Richard A. Adegbola Bill & Melinda Gates Foundation, Seattle, Washington, U.S.A.; and formally Medical Research Council (U.K.) Laboratories, The Gambia

Samba O. Sow Centre pour le De´veloppement des Vaccins-Mali (CVD-Mali), Bamako, Mali

J. Anthony G. Scott KEMRI Wellcome Trust Research Programme, Kilifi, Kenya; and Nuffield Department of Clinical Medicine, University of Oxford, Oxford, U.K.

INTRODUCTION Protein-conjugated Haemophilus influenzae type b (Hib) vaccines represent a scientific and technological triumph over one of the most widespread and pernicious infant diseases of the 20th century. The public health triumph, however, lags further behind. Each year Hib causes approximately 3 million cases of meningitis and severe pneumonia worldwide leading to 386,000 deaths in children aged less than five years (1). Of those who survive meningitis, 30% to 40% go on to develop lifelong disabilities such as mental retardation or hearing loss (1). Conjugation of Hib polysaccharide to immunogenic proteins overcomes the innate deficiencies of young infants in recognizing repeating carbohydrate antigens, and this discovery has led to the development of commercial vaccines that have almost eliminated Hib disease from the industrialized world. In 2000, a decade after the efficacy of Hib conjugate vaccines had been established, the introduction of vaccine was so limited that less than 2% of the global disease burden was being prevented (2). In 2001, the global alliance for vaccines and immunization (GAVI) offered financial support to developing countries to introduce Hib conjugate vaccine into routine childhood immunization over five years (3). This improved global vaccine coverage substantially, but in 2006, the proportion of the world’s children receiving Hib vaccine was still only one quarter. Hib is low on the priority list of most developing countries. It is a fastidious organism that is difficult to culture and is therefore rarely seen in settings with limited hospital and laboratory resources. In the perception of the medical profession and of the public in developing countries, it is almost invisible. Any claim to public health importance was obscured by the noisy clamor of advocacy groups for HIV, tuberculosis, and malaria. In 2005, GAVI funded a multistakeholder group, ‘‘The Hib Initiative,’’ to assist countries to evaluate the local burden of Hib disease, to assess the cost-effectiveness of Hib vaccine in their countries, and to formulate financial plans to integrate the vaccine into the routine childhood immunization program (4).

GAVI also extended its financial support for vaccine introduction by a further five years. In 2006, the World Health Organization (WHO) published a position statement recommending that Hib vaccine be included in all routine childhood immunization programs, even in the absence of local disease burden data (1). As a consequence of these three steps, many more countries made applications in 2007 to introduce Hib vaccine into routine immunization programs, and it is likely that the global burden of Hib morbidity and mortality will be substantially reduced by 2012.

EFFICACY OF PROTEIN-CONJUGATED Hib VACCINE IN DEVELOPING COUNTRIES Commercial vaccines against Hib in the 1970s used purified bacterial capsular polysaccharide, polyribosylribitol phosphate (PRP), as antigen. These were not immunogenic among children aged less than 18 months, who were the primary risk group. They did not induce booster responses and had no effect on nasopharyngeal carriage, but they were licensed and used for over a decade in children aged more than 24 months in industrialized countries. Conjugation of PRP to immunogenic proteins was the key scientific insight that overcame these deficiencies by converting the infant immune reaction to PRP from a T cell–independent response to a T cell–dependent response. The principle that conjugation of proteins to polysaccharides could enhance the immunogenicity of the polysaccharide antigen had been demonstrated in 1929 in rabbits using pneumococcal capsular polysaccharides antigens (5). Fifty years later, PRP was successfully conjugated variously to diphtheria toxoid (PRP-D), mutant diphtheria toxoid (PRP-CRM, HbOC), the outer membrane protein of Neisseria meningitidis (PRPOMP), and tetanus toxoid (PRP-T). When administered to infants two to six months old in the industrialized world, the efficacy of each of these conjugates was more than 94% (6–8). Studies of native American populations provided conflicting evidence about the likely efficacy of conjugate Hib vaccines in

Chapter 44: Hib and Infant Immunization in Developing Countries

cultural and socioeconomic settings more akin to developing countries. The efficacy of PRP-OMP against invasive Hib disease among Navajo infants was 95%, and the protection began with the first dose administered (7). At the same time, PRP-D administered to Alaskan native infants scarcely raised antiPRP antibody concentrations after two doses and did not protect significantly against invasive disease (9). Nonetheless, protein conjugate Hib vaccines were licensed for infant use in 1990, and with widespread introduction, invasive Hib disease has almost disappeared from the industrialized world (10,11). To achieve similar success in the developing world appeared more complex. The incidence of Hib disease and the prevalence of nasopharyngeal carriage were higher in developing countries, and the disease occurred in younger children, with half of all cases occurring before the age of eight months (12,13). In addition, Hib was a significant cause of pneumonia in developing countries, and it was unclear whether the systemic antibodies induced by vaccine would diffuse sufficiently into the lung to prevent cases of pneumonia, particularly those that came about by direct spread of the pathogen from the upper to the lower respiratory tract. The first evidence of postlicensure vaccine effectiveness outside the industrialized world came from Chile, where existing epidemiological studies had confirmed a significant burden of Hib disease (14). As part of a strategy to determine country-specific policy toward the new conjugate vaccines, the 71 vaccine health centers of Santiago city were divided into two approximately equal groups and randomly assigned to administer either DTP (diphtheria-tetanus-pertussis) vaccine alone or DTP with PRP-T in the routine immunization schedule. National census data were used to define the populations served by the two sets of health centers and the numbers of cases of invasive Hib disease occurring in the two sets of areas were compared to establish vaccine effectiveness. Approximately 46,000 infants were served with vaccines by each of the two health center sets. The vaccine effectiveness was 90.2% (95% CI, 74.5–100%) for invasive Hib disease, and it did not vary by age at onset of disease. The effectiveness against Hib pneumonia (80%) was lower than that against meningitis or other invasive Hib syndromes (91–100%) but the number of culture-proven pneumonia cases was small (15). The first randomized controlled efficacy evaluation of PRP-T was conducted in The Gambia between March 1993 and March 1996 (16). The only previous evaluation of PRP-T was an open study comparing the incidence of Hib disease in four districts of Oxfordshire, United Kingdom, where PRP-T had been offered simultaneously with DTP, against the incidence in four districts where PRP-T had not been offered (8). In The Gambia, 42,848 infants were randomly assigned to receive either PRP-T mixed with DTP or DTP alone. PRP-T was given on three occasions at a median age of 11 weeks, 18 weeks, and 24 weeks. The primary endpoints of the study were (i) protection against proven Hib pneumonia after two or three doses of vaccine and (ii) protection against all invasive Hib disease after three doses of vaccine. There were 10 eligible cases of proven Hib pneumonia among the controls and none among the vaccinees who had received two or three doses, giving a vaccine efficacy of 100% (95% CI, 55–100%). There were 19 eligible cases of invasive Hib disease among the controls and one among the vaccinees who were fully vaccinated giving a vaccine efficacy of 95% (95% CI, 67–100%). The point estimate of efficacy against invasive disease after one dose of vaccine was 44%, but this had little precision (95% CI, 85%, 85%).

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On the basis of culture of blood and lung aspirate material, several previous studies of pneumonia etiology in The Gambia had suggested that Hib was responsible for approximately 7% of cases of severe pneumonia (17–20). The estimate of vaccine efficacy was also obtained predominantly from cases of pneumonia with blood cultures positive for Hib, although in two cases Hib was isolated from lung aspirate cultures. However, for the first time the question of nonbacteremic Hib pneumonia was also examined in detail in this study (16). In the trial, 1821 episodes of pneumonia were investigated among study participants, and the protective efficacy of the vaccine was estimated for different case definitions irrespective of confirmatory etiologic data. Using the sensitive but poorly specific WHO-defined clinical criteria for pneumonia (cough with fast breathing or lower chest wall indrawing), vaccine efficacy was 4.4%; among the subset who required admission to hospital (with lower chest wall indrawing), the vaccine efficacy was 6.5%. Neither of these estimates could be distinguished statistically from a null effect. However, the vaccine efficacy against radiologically defined pneumonia was 21.1% (95% CI, 4.6–35%) and against radiologically confirmed lobar pneumonia or pneumonia with effusion it was 25.2% (95% CI, 0.24–44%) (16). For a clinically defined endpoint like pneumonia, which can be caused by numerous different etiologic agents, the observed vaccine efficacy estimates in themselves do not define any precise biological parameter. Rather, they help to establish limits to the parameters of which they are composed. The observed vaccine efficacy is equal to the product of two measures: (i) the true vaccine efficacy against all Hib pneumonia and (ii) the proportion of all cases meeting the clinical case definition that have been caused by Hib. If the true vaccine efficacy against Hib pneumonia is 100%, that would imply that 21.1% of all radiologically defined pneumonia cases are caused by Hib. Equally, if the true efficacy of the vaccine against Hib pneumonia is closer to the observed estimate (0.80) among bacteremic Hib pneumonia patients, this would imply that 26.4% (21.1/0.8) of all radiologically defined pneumonia is caused by Hib. These deductions suggest that Hib contributes considerably more to the etiology of severe pneumonia than had previously been anticipated. The analytical approach of using vaccine as a ‘‘probe’’ to estimate disease burden has become a useful tool in Hib and pneumococcal epidemiology. For example, to test whether Hib causes *20% of radiologically confirmed pneumonia in other populations, the clinical data and radiographs of the Chilean PRP-T effectiveness study were reexamined. The incidence of radiologically confirmed pneumonia was calculated for children aged 4 to 23 months who received at least two doses of conjugate Hib vaccine and compared against the incidence among children from control areas. Clinical and radiological confirmations were abstracted from the in-patient records of children admitted to the three public hospitals in the area on the basis of a screen of discharge diagnoses. If the radiological report was insufficiently precise to distinguish pneumonia, the original radiograph was retrieved and classified by a radiologist blinded to the child’s vaccine status. Vaccine status was inferred from residence in one of the vaccine intervention areas or one of the control areas. Although the study size was inadequate to define precisely the efficacy of vaccine against radiologically confirmed pneumonia (consolidation or effusion), the point estimate of 22% (95% CI, 7%, 43%) was very similar to that obtained in The Gambia trial (21).

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A prospective vaccine probe study in Asia was conducted in Lombok, Indonesia, in a trial designed specifically to estimate the incidences of vaccine preventable pneumonia and meningitis attributable to Hib in children aged less than two years (22). In a cluster-randomized design, undertaken between 1998 and 2002, 55,073 children were immunized with either Hib conjugate vaccine combined with DTP vaccine (DTPPRP-T) or with DTP alone. There were 818 hamlets in the study area, which served as units of randomization. The burden of Hib disease within each syndrome category was estimated as the incidence rate difference between the two groups, defining Hib vaccinated children as those who received at least one dose of Hib vaccine. The incidence of Hib vaccine preventable meningitis, defined as a cerebrospinal fluid (CSF) leucocyte count of more than 10  106/L, was 67/100,000 child years (95% CI, 22–112%), confirming that Hib meningitis occurs with a similar frequency in Indonesia as in most settings in Africa, Europe, and the Americas. For pneumonia, defined as either severe clinical pneumonia, pneumonia admitted to hospital, or radiologically confirmed pneumonia, there was no significant difference in the incidence rates among vaccinated and unvaccinated children. For ‘‘clinical pneumonia,’’ which includes nonsevere pneumonia defined by WHO as a raised respiratory rate, the incidence of Hib vaccine preventable disease was 1561/100,000 child years (95% CI, 270–2853%). Nonsevere pneumonia was common in this setting with a background incidence among controls of 4 episodes for every 10 children each year; Hib vaccine prevented 4% of such episodes (22). The effectiveness of PRP-T Hib vaccines against radiologically confirmed pneumonia has also been examined in three casecontrol studies (23–25). During introduction of conjugate Hib vaccine in Brazil, the immunization histories of 427 cases of radiologically confirmed pneumonia aged less than two years were compared with 854 age- and location-matched controls. After adjusting for other covariates, the estimated effectiveness of two or more doses of vaccine was 31% (95% CI, 9%, 57%) (23). This division of vaccine doses classifies children who have received a single dose as unimmunized and, assuming that one dose has at least some protection against pneumonia, it would bias the effectiveness estimate of immunization downward. In a second study from Colombia, with a very similar design, 389 cases of radiologically confirmed pneumonia were compared with 774 age-, sex-, and socioeconomically matched controls randomly selected from among children attending child health clinics at the hospitals where the cases were admitted. Several exposure variables were associated with vaccination status including birth weight, maternal gestation less than nine months, breastfeeding, day care attendance, prior hospitalization, smokers in the household, cooking in the sleeping room, dwelling type, social security scheme, and parental education. Adjusting for all significant covariates, the effectiveness of one, two, and three doses of Hib vaccine was 47% (95% CI, 2–72%), 52% (3–76%) and 52% (7–78%), respectively (24). In Bangladesh, pneumonia in children less than two years was defined by radiological interpretations of either three study radiologists or two WHO radiologists among 475 or 675 children, respectively. Both sets of radiologists agreed upon evidence of pneumonia in 343 children. Four healthy controls per case were recruited from the community and matched on age, sex, season, and distance from hospital, and two controls per case were recruited among hospitalized children who were residents of the study area and were admitted with a diagnosis other than pneumonia or meningitis. Where both sets of radiologists agreed upon the diagnosis of pneumonia,

the adjusted vaccine effectiveness for at least two doses was 34% (95% CI, 6–53%) in the community case-control study and 44% (95% CI, 20–61%) in the hospital case-control study. Using the case definitions of pneumonia based on the WHO radiographers alone, the vaccine effectiveness estimates were 17% (95% CI, 10%, 38%) and 34% (95% CI, 14–49%), respectively. There are three points of interpretation in the case-control studies. First, the precision of the vaccine efficacy estimates is poor; all the studies had wide confidence intervals. Second, they are susceptible to selection bias, yielding different results depending on the mode of control selection. Third, they highlight the problem of interobserver variation in the interpretation of radiographs in pneumonia. Following the randomized controlled efficacy trial in The Gambia, the WHO set out a methodology for standardizing radiographic interpretation based on a common vocabulary and a demonstration set of typical radiographs with agreed interpretations (26). Using this standard, readers have achieved moderately good interobserver agreement (27), and it has been used successfully in the evaluation of pneumococcal conjugate vaccines (28). Despite the evident limitations noted, the evidence from both trial designs and case-control studies using a variety of different radiological standards, suggests that protein conjugate Hib vaccines are highly effective against Hib pneumonia and that the pathogen is the likely etiologic agent in between 20% and 50% of episodes of radiologically confirmed pneumonia in children aged less than two years in developing countries. The exception to this generalization is the Indonesia vaccine probe study, where the burden of clinical pneumonia that was prevented by Hib vaccine was 1561/100,000 person years of observation, but this accounted for only 4% of clinical pneumonia cases; furthermore, there was no suggestion that this proportion increased with increasing severity of pneumonia classification (22). Finally, the efficacy of Hib conjugate vaccine in HIV has been evaluated during the preparations for a randomized controlled trial of conjugate pneumococcal vaccine in South Africa. Hib vaccine was introduced for all children at the study site in March 1998, and the incidence of culture-proven invasive Hib disease after vaccine introduction (March 1998–June 1999) was compared against the incidence prior to vaccine introduction (January–December 1997) to derive vaccine effectiveness estimates. On the basis of the known prevalence of HIV infection among adults and assumptions about maternalto-child HIV transmission in the site, HIV positive and negative denominator populations were imputed. There were changes in disease surveillance in March 1997, which may have increased case ascertainment and slightly reduced the estimated efficacies. Among children who successfully received all three doses, the effectiveness of Hib vaccine against invasive disease was 43.9% (95% CI, 76.1%, 82.1%) in those with HIV infection and 96.5% (95% CI, 74.4–99.5%) in those without (29). Hib vaccine was significantly less effective in HIV-positive children than in HIV-negative children, but it did not appear to be harmful. In settings where the seroprevalence of HIV is high, the rationale for immunization remains strong because of the potent protective effect among HIV-uninfected children.

Hib DISEASE BURDEN IN DEVELOPING COUNTRIES Measuring the burden of Hib disease can be particularly difficult in developing countries where routine systematic surveillance for bacterial diseases is usually not in place

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because the expertise and facilities for clinical and laboratory diagnosis of Hib infections are inadequate. However, innovative methods have been developed to define Hib disease burden, and these have proven useful for making informed decisions about vaccine introduction and impact evaluation in these countries.

Hospital and Community-Based Surveillance for Hib Disease Hospital-based surveillance for bacterial meningitis defines the proportion of children admitted with Hib meningitis at a particular hospital for a given period of time. This must be based on documented definitions, clinical and laboratory investigations including a record of history and, when possible, a biological test for prior antibiotic use. Incidence of Hib meningitis cannot normally be calculated because the patient catchment population is usually difficult to define. When the hospital facility is contained within a well-defined catchment population, the incidence of Hib disease, particularly of meningitis, can be estimated after adjustment for access to care. The comparability of surveillance data in different areas is dependent on consistency in patient selection and enrolment, and laboratory investigations to ensure that eligible patients are captured and investigated appropriately. A generic protocol was developed by WHO in 1996 for in-country assessment of disease burden for Hib meningitis (30). The protocol contains guidelines for selection of surveillance sites and information on clinical, laboratory, and epidemiological methods, and has been made accessible electronically (31). The main limitation of this method is that Hib disease due to pneumonia cannot be adequately measured, and in many developing countries a significant proportion of meningitis cases die at home without accessing care. Because the syndromes of clinical meningitis and pneumonia are not etiology specific, clinical findings alone are not sufficient to diagnose Hib disease. Therefore, clinical suspicion of meningitis or pneumonia must be accompanied by collection of appropriate specimens such as CSF and blood, respectively, for laboratory isolation of the bacterium or detection of bacterial antigens using latex agglutination tests. Polymerase chain reaction (PCR) methods can be used when available for direct detection of Hib in CSF. In contrast, the confirmatory diagnosis of Hib pneumonia is more problematic. Available clinical and radiological methods lack sensitivity or specificity. Although laboratory isolation of Hib from blood of patients with clinical evidence of pneumonia can be definitive, the sensitivity is poor and the level of expertise required is often lacking in many developing countries (32,33). For these reasons, surveillance for Hib disease is often targeted at meningitis alone.

Population-Based Surveillance Despite its limitations, blood culture surveillance for invasive bacterial disease has shown that Hib is a major cause of bacteremia in children in Africa. Population-based studies have been used to document Hib disease burden as a major cause of morbidity and mortality in several countries including Kenya, Mali, and The Gambia. At a district hospital located in an area with a well-defined catchment population on the coast of Kenya, acute bacterial infections were a common cause of admission to the hospital. Blood was cultured from 19,339 patients on admission to hospital between 1998 and 2002. Hib accounted for 12% (136 of 1132) of bacterial isolates from among children aged less than five years, and for 19% (18 of

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103) of isolates from among children who died in hospital on the day of admission (34). The annual incidence of Hib bacteremia was 159, 120, and 60 per 100,000 children aged less than one year, two years, and five years, respectively. The incidence of acute bacterial infections caused by Hib was eclipsed only by those of pneumococcus and nontyphoidal Salmonella in children less than five years. In children aged two months to one year, Hib was of the same rank with pneumococcus among bacterial species isolated from patients with bacteremia (34). Similarly, in Mali, the age-specific incidence of Hib disease was determined after the establishment of a bacteriology laboratory in the main hospital serving Bamako, the capital city. The study investigated 3592 children admitted to the hospital over a two-year period with suspected invasive bacterial disease. Hib was isolated from 207 children; 98% of these were aged less than five years, and 60% (124) were meningitis cases. The annual incidence of Hib disease per 100,000 child years was 45 among children less than five years, and 158 among children aged less than one year. The peak incidence, 370/100,000 person years, was found among children aged six to seven months, and 12 (57%) of 21 recorded Hib deaths also occurred in this age group (35). As noted earlier, the incidence of Hib meningitis was high in The Gambia prior to vaccination, with a rate of 297/100,000 per year among children aged less than one year and 60/100,000 among children less than five years (12). The disease occurred mainly in younger children; 84% of cases were aged less than 12 months and 45% were less than six months. Neurological sequelae were common. The case fatality rate was 30%, and only 45% recovered completely from Hib meningitis (36). Pneumonia was more common and its outcome was worse in Gambian children than in the developed countries. In common with most of the other developing countries, epiglottitis was rarely seen (37).

Other Approaches to Disease Burden Assessment The Hib rapid assessment tool (Hib RAT) was developed by WHO to estimate the local burden of Hib disease and mortality where population-based estimates are not available (38). The tool uses two complementary methods to achieve these objectives. The first method uses routine hospital meningitis data within a well-defined population to estimate the local incidence of Hib meningitis, then inflates this figure by a factor of five to estimate Hib pneumonia incidence and extrapolates these rates across the country. The second method uses the under-five mortality rate, if known, to calculate the number of deaths caused by acute respiratory infections, and then estimates that 13% of these deaths are caused by Hib. A Hib RAT is usually completed in 7 to 10 days by a team of two to three local health officials and one to two consultants. The accuracy of the Hib RAT is dependent on the quality of local Hib disease data. In Africa and some countries of the Middle East, Hib RAT meningitis incidence rates have been similar to estimates from population-based studies in these regions. In contrast, variable Hib RAT estimates have been obtained from Eastern Europe and Asia, where Hib meningitis appears to be less well defined (38). Despite its limitations, the Hib RAT has been used by several countries in Africa to provide baseline data on Hib disease prior to vaccine introduction. The Hib RAT manual, including calculation worksheets, is available from WHO (39). Sentinel site surveillance was established for Hib meningitis by the WHO pediatric bacterial meningitis surveillance

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network (AFRO-PBM) in 2001. One site was selected per country in 26 countries in Africa. AFRO-PBM data has been used in several countries to evaluate impact following Hib vaccination (40). This approach has been expanded by several regional surveillance networks such as netSPEAR in East Africa (41), with support from the PneumoADIP (GAVI’s Accelerated Development and Introduction Plan for Pneumococcal vaccines).

IMPACT OF PROGRAMMATIC INFANT IMMUNIZATION IN DEVELOPING COUNTRIES Although the uptake of vaccination has been slower in developing countries than was initially expected, there have been dramatic reductions in the incidence of Hib disease wherever the vaccine has been used in infant immunization programs. The protective impact of Hib immunization has been particularly remarkable in poor developing countries despite less than optimal vaccine coverage and irregular vaccine supply (42) and also in settings with a high prevalence of HIV infection (43). Demonstration of vaccine efficacy during trial conditions is not always a guarantee that it will be effective in routine use in developing countries because suboptimal cold-chain and transport conditions can reduce vaccine effectiveness. In contrast, additional benefits of Hib vaccination from herd immunity mediated by reduced oropharyngeal carriage can make vaccination against Hib disease more effective than is suggested by an efficacy trial.

The Gambia Hib vaccine was introduced into The Gambia Expanded Programme of Immunization (EPI) in May 1997 after appropriate training and an awareness campaign. Vaccination was made possible through a five-year donation by the vaccine manufacturers Pasteur Merieux (Sanofi Pasteur, Lyon, France). From May 1997 to April 2002, a study was undertaken to evaluate the effectiveness of the Hib vaccine against meningitis, to estimate the herd immunity effect as shown by reduced oropharyngeal carriage of Hib and to ascertain whether Hib disease would occur more frequently or in an atypical form among older children. A standardized protocol was used to carry out surveillance for meningitis and other Hib diseases in the same population where the efficacy trial was undertaken. The vaccine efficacy was estimated from a nested case-control study. Vaccine supply was irregular during the period and vaccine coverage was only *70%. The annual incidence of Hib meningitis dropped from 200/100,000 to 21/100,000 among children aged less than one year during the first two years of surveillance (44). It dropped further to 0 in infants and from 60 to 0/100,000 in children aged less than five years (Fig. 1) by the end of surveillance in 2002 (42). Among 49 children identified as definite cases of invasive Hib disease, 28 (57%) had not been vaccinated, 17 (35%) had received only one dose, two (4%) had received two doses, and two (4%) had received three doses. No cases of epiglottitis were observed. Introduction of routine Hib immunization did not lead to the emergence of unexpected forms of Hib disease in older children in The Gambia. Notably, the prevalence of Hib carriage decreased from 12% to 0.25% (p < 0.0001), and only two doses of vaccine were required for direct protection against invasive Hib disease (vaccine efficacy 94%; 95% CI, 62–99%). Although Hib disease was eliminated in The Gambia by 2002, five new cases were detected by informal surveillance

Figure 1 Incidence of Hib meningitis per 100,000 per year, in children less than five years of age in western region of The Gambia (dotted lines: pointwise 90% likelihood-based confidence limits). Source: From Ref. 42.

between July 2005 and April 2006 (45). These children were aged 3 to 36 months, three of them had meningitis and two had septicemia. The cause for this reemergence is not clear, but waning immunity, deterioration of vaccine handling, delivery and coverage, or the emergence of a hypervirulent strain have all been suggested as explanations. It emphasizes the importance of continued bacteriological surveillance in developing countries after vaccine introduction.

Kenya Prior to the introduction of routine immunization with Hib conjugate vaccine in Kenya, the median age of invasive Hib disease was eight months, and 82% of cases were younger than two years (13). The annual incidence of Hib invasive disease in children aged less than five years was 66/100,000, which is similar to the incidence recorded in several other African countries such as South Africa (47/100,000) (46) and Mali (45/100,000) (35). A pentavalent vaccine containing combined DTP/hepatitis B/conjugate Hib was introduced into the Kenya EPI in November 2001, with three doses given at ages 6, 10, and 14 weeks. A booster dose was not administered. The effectiveness of vaccine introduction was evaluated between 2000 and 2005 in one district, Kilifi, using a combination of hospitalbased surveillance and a population register from an associated Demographic Surveillance Study (DSS), covering a population of 38,000 children aged less than five years. The incidence of culture-proven Hib invasive disease before and after the introduction of the immunization program was compared to determine vaccination program effectiveness. The annual incidence of invasive Hib disease in children aged less than five years declined from 66/100,000 in the year before the vaccine introduction to 7.6/100,000 three years after introduction (Fig. 2). In the last year of the study, vaccine effectiveness was 88% (95% CI, 73–96%) among children aged less than five years and 87% (95% CI, 66–96%) in children aged less than two years (13). By extrapolation it was estimated that the vaccine prevented 3370 hospitalizations with culture-proven invasive

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Serotype Replacement Disease Serotype replacement disease, which is well documented following introduction of conjugate pneumococcal vaccine in the United States (47), has not been observed following Hib vaccine introduction in developing countries. Early reports from the National Public Health surveillance in Brazil suggested an increase in the annual incidence of H. influenzae type a meningitis in the first year after vaccine introduction from 0.01 to 0.14/100,000 total population (48). In the subsequent three years, the incidence returned to pre-vaccine levels, and the transient increase is probably best explained by heightened surveillance in the aftermath of the vaccine introduction.

Herd Protection Effect

Figure 2 Invasive Hib disease in children aged less than five years in Kilifi DSS.

Hib disease in children aged less than five years in Kenya in that year. In contrast to The Gambia, where a significant drop in incidence of Hib disease was recorded two years after introduction of the vaccination program, incidence of Hib disease did not decrease significantly in Kenya until the third year after vaccine introduction. As in The Gambia, there was no catch-up campaign of older children when Hib vaccine was introduced into Kenya’s EPI. It is important to take vaccination schedule and years of surveillance before and after introduction of vaccination into consideration when evaluating the impact of routine use of a new vaccine in a developing country setting.

Other Developing Countries In Malawi, routine immunization with Hib vaccine was introduced into EPI in a pentavalent formulation in January 2002. The vaccination schedule consisted of three doses given to infants at ages 6, 10, and 14 weeks. A booster dose was not given. Surveillance was undertaken with a focus on meningitis using the AFRO-PBM database, and was located at Blantyre district covering a population of about 1 million. A casecontrol method was used to estimate vaccine effectiveness using children hospitalized with Streptococcus pneumoniae meningitis as controls. The frequency of Hib meningitis admissions began to decrease only one month after vaccine introduction. Nine months after introduction, the number of Hib meningitis cases had dropped to only two to three per month from a baseline of 12 or more. Cases of Hib meningitis occurred only in children who were not fully vaccinated. The frequency of presentations of pneumococcal meningitis remained constant indicating that the observed results were not due to declining laboratory performance. Additionally, Hib disease decreased in older, unvaccinated children after the first year of the program, which is suggestive of an indirect effect of vaccination. High vaccine effectiveness for Hib meningitis was demonstrated among children aged less than five years (94%; 95% CI, 70–99%). This occurred despite limited health care resources and a high burden of HIV infection (40). At least 14% of children with Hib meningitis were coinfected with HIV in the study population.

Hib conjugate vaccines reduce asymptomatic nasopharyngeal carriage of Hib in vaccinated children, leading to a reduction in transmission with subsequent herd protection effect. In The Gambia, Hib disease was eliminated in the community at a point when, based on coverage estimates, only 41% of potential cases would have been protected by the direct effects of immunization (42). Although two doses were required for the direct protection of children in the Gambia study, most received their second dose too late to benefit from direct protection. The latter doses may be required to achieve and maintain the high levels of antibodies required for protection against Hib carriage. Nasopharyngeal carriage studies may therefore be helpful in surveillance programs to observe indirect vaccine effect after Hib vaccine introduction.

COST-EFFECTIVENESS OF CONJUGATE Hib VACCINES IN DEVELOPING COUNTRIES An assessment of the costs and benefits of introducing new vaccines is critical in the developing world, where many competing initiatives vie for spending from very limited health budgets. In March 2007, GAVI detailed the second phase of vaccine support for 72 eligible developing countries—those with a gross domestic product per capita of less than $1000 in the year 2005. Developing countries will be required to co-pay for vaccine at a subsidized price, and the level of co-payment is decreased for each new vaccine introduced. Assuming that conjugate Hib vaccine is the first new vaccine (usually in a formulation with DPT and hepatitis B vaccines), countries introducing this vaccine will be asked to contribute $0.30/dose. The questions that drive present cost-effectiveness analyses are therefore, (i) What is the ratio of vaccine introduction costs to the sum of treatment (and societal) costs that would be averted by vaccine introduction? (ii) On the assumption that countries will eventually have to sustain their health services independently and pay the full economic price for vaccine, what happens to the cost-benefit ratio as the cost per dose increases from its presently subsidized level? The first approach to these questions from Africa was a cost-benefit study from South Africa in 1995 (49). The authors used an estimate of the incidence of invasive Hib disease, 133 cases/100,000 children aged less than five years, which is comparable with several other centers in Africa. They converted the value of a life into monetary terms to quantify the benefits of the vaccine program. Despite assuming a vaccine price of US$14 per dose, the authors concluded that conjugate Hib vaccine was cost saving from a societal perspective. Three studies from newly industrializing countries, Chile, Malaysia,

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and The Philippines, have also concluded that Hib vaccine is cost saving from the public health provider perspective using vaccine cost assumptions of $0.30, $4.30, and RM19.55 (in 1999) (50–52). In each case, calculation of the benefits of vaccination was restricted to quantification of meningitis cases prevented with no reference to the large anticipated gains against Hib pneumonia. In Kenya, an incremental cost-effectiveness model was developed around the national birth cohort in 2004 followed for five years (53). Total healthcare costs were compared using the scenario of pentavalent vaccine introduction (conjugate Hib– hepatitis B–DTP) against the status quo (DTP alone). Treatment cost data from the government perspective were obtained directly from observations of cases of pneumonia and meningitis in Kenyan hospitals, and household costs were not included in the analysis. Future costs and outcomes were discounted 3% per year. Hib disease incidence and case fatality estimates were obtained from published data from Kenya (34) and adjusted using assumptions about access to care. The disease burden model incorporated three syndromes, meningitis, pneumonia, and nonmeningitis invasive disease, and the estimate of pneumonia was derived by applying a ratio of 5:1 in cases of pneumonia to meningitis (33). Using the prevailing cost of pentavalent vaccine (US$3.65), the cost per discounted DALY (disability-adjusted life year) was $38 (95% CI, 26–63), and per death averted was US$1197 (95% CI, 814–2021%). The WHO suggests that an intervention may be considered cost-effective if the costs per discounted DALY averted are less than the per capita GDP (54) of the country. Therefore, for Kenya, with a per capita GDP of US$481 in 2004, the vaccine is considered highly cost-effective. The cost-effectiveness ratios were most sensitive to changes in the cost of pentavalent vaccine, and at a per dose cost of less than US$1.82, the intervention would become cost saving. A similar approach was taken to estimate the incremental cost-effectiveness of Hib vaccine in Indonesia, using the 2005 birth cohort and burden of disease and vaccine efficacy data derived from the Hib vaccine probe study in Lombok (55). Taking the UNICEF supplied cost per dose of $2.27 the cost per DALY averted was £67. Taking the GAVI-subsidized cost per dose ($0.37), it was estimated that the vaccine program would save $3.7 million.

PROBLEMS IMPEDING INTRODUCTION OF Hib VACCINE INTO DEVELOPING COUNTRIES Between licensure in 1990 and the advent of GAVI support in 2000, the obvious impediment to Hib vaccine introduction in the developing world was cost. GAVI has provided support to 23 countries (15 in Africa) over the last seven years and has committed to continue supporting vaccine introduction up to 2015. In the long term, competition among manufacturers should drive prices down to a point where they become affordable directly to developing countries. Encouragingly, vaccine may also be manufactured in the developing world: the Serum Institute of India has recently obtained a license to manufacture Hib vaccine in collaboration with the Netherlands Vaccine Institute, and a number of other developing country producers have Hib containing combination vaccines under development. Cost was also the driving force behind the evaluation of fractional doses of vaccine. In South Africa, the immunogenicity of one-tenth of the dose of PRP-T was noted to be equivalent to that following the full dose (56). Studies of other dilutions of

PRP-T at one-half, one-quarter, and one-eighth dose have confirmed the immunogenicity of fractional doses and shown that the antibodies stimulated by fractional doses are of similar or greater avidity (57,58). Although this approach is potentially economical, there are considerable obstacles to the practical use of fractional doses: We have no evidence of clinical protection; the duration of protection is not known; the products are not licensed at these doses; and there are considerable practical problems in implementing a schedule that requires dose splitting in the field. Fractional doses are not a realistic solution within the foreseeable future, but it is possible they may be implemented if developing countries begin to take on direct financial responsibility for introduction of Hib vaccination themselves (59). However, cost is not the only impediment to Hib vaccine introduction. In many areas, there are continuing uncertainties about the magnitude of the Hib disease burden and the costeffectiveness of vaccine introduction. Even where good data exist, these are not always given credence and may not influence policy. The burden of disease and the effectiveness of vaccine need to be communicated directly to opinion leaders and policy makers to have an impact on decision making. In Mali, Hib vaccine was not introduced until the data on disease burden were communicated directly to the president (35). Furthermore, many countries are worried about the programmatic implementations of adding additional antigens to their existing childhood immunization schedule, especially where immunization coverage is poor. Many immunization program managers were reluctant to introduce Hib in a second injection at each vaccine visit for fear that it would reduce compliance with later doses in the childhood schedule. Hib conjugate vaccine is now available in combination with the major antigens of childhood immunization programs (DPT and hepatitis B), which can be given in a single pentavalent vaccine injection. A further anxiety is the sustained effectiveness of the vaccine program in the developing world. The primary immunization schedule for Hib vaccine is three doses given at 6–10–14 weeks or 2–4–6 months. In most developed countries, a booster dose is also given in the second year of life. In the United Kingdom, Hib disease was controlled using a three-dose primary schedule and no booster, though vaccine introduction was accompanied by a catch-up campaign targeting all children under the age of four years. Seven years after introduction, there was a resurgence of Hib disease (60,61), which was attributed to waning individual concentrations of antibody with age leading to inadequate population immunity. In response, a booster dose was introduced into the U.K. schedule at 15 months of age. If a booster dose is ultimately required in developing world schedules, this would require a radical reconfiguration of the present EPI, which currently targets children only in the first year of life, or the introduction of a Hib booster dose at the same time as measles vaccination, which is given toward the end of the first year of life.

CONCLUSION Hib meningitis, pneumonia, and septicemia are common, serious infections of infants and young children throughout the developing world. Conjugate Hib vaccines are highly effective at protecting the individual child and, by reducing Hib carriage among vaccinees, they also protect the whole community. Developing countries that have introduced Hib vaccine have almost eradicated Hib disease. Spreading the coverage of Hib vaccine throughout the developing world will reduce infant

Chapter 44: Hib and Infant Immunization in Developing Countries

Figure 3 Status of Hib vaccine in Africa, WHO database, May 2007.

mortality and improve child health by reducing significant infant morbidity and the devastating long-term sequelae of meningitis. Encouraging progress has been made introducing Hib vaccine into routine immunization programs in developing countries. The WHO position statement—that Hib vaccine should be included in all routine childhood immunization programs, even in the absence of local disease burden data (1)—has encouraged further uptake. In Africa, where the greatest burden of Hib disease lies, all but one country has now applied to GAVI for support for Hib vaccine introduction (Fig. 3). The challenge for the future is to sustain Hib vaccine within routine childhood immunization programs. In the short term, this will require good disease surveillance, evaluation of cost-effectiveness, and communication to policy makers. In the long term, it will require a reduction in vaccine costs allowing countries to purchase Hib vaccine independent of GAVI funding. At stake are the lives of nearly 4 million children over the coming decade.

ACKNOWLEDGMENT We thank Prof Kim Mulholland for his critical comments of an earlier draft of this chapter.

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45 Pneumococcal Protein-Polysaccharide Conjugate Vaccines Cynthia G. Whitney Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

Orin S. Levine Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A.

EPIDEMIOLOGY OF PNEUMOCOCCAL DISEASE AND CARRIAGE Streptococcus pneumoniae is a major cause of pneumonia, meningitis, bloodstream infections, and acute otitis media. Disease rates are highest in children younger than five years, are lower in older children and healthy young adults, and increase again in the elderly (1). Pneumococcal disease is endemic worldwide. Generally, rates of disease, deaths, and complications are higher in developing countries than in industrialized settings, but artifactual differences in disease risk are also observed because of variation in the use of diagnostic tests, particularly blood cultures. Case fatality can be high for invasive pneumococcal infections, ranging up to 20% for sepsis and 50% for meningitis in developing countries. Among meningitis survivors, long-term neurologic sequelae such as hearing loss, mental retardation, motor abnormalities, and seizures can occur in up to 58% of cases, as seen in the Gambia (2). Major risk factors for infection include age less than two years, underlying immunodeficiency (including HIV/AIDS), sickle cell disease, certain other chronic illnesses, day care attendance, and exposure to tobacco smoke; breastfeeding has been shown to be protective (3). Risk of infection can also vary with race. Genetic factors (e.g., sickle cell disease) or cultural practices (e.g., premastification of food for infants) may be involved but associations with race are hard to distinguish from socioeconomic or environmental risk factors that may also correlate with race. Invasive disease rates are higher among indigenous populations of Australia and New Zealand and among the black, Alaska Native, and American Indian populations in the United States relative to the general population (4–6). Adults who have frequent exposure to young children are also at elevated risk for pneumococcal infections (7). For both children and adults, preceding viral infections such as influenza can lead to secondary pneumococcal infections. Ninety-one distinct serotypes of S. pneumoniae have been identified on the basis of structural differences in the polysaccharide capsule, but not all serotypes are equally capable of causing disease. Approximately 10 or 11 serotypes account for at least 70% of invasive pediatric infections in all regions of the world (8). The distribution of serotypes causing disease varies by age, disease syndrome, disease severity, geographic region, and over time. Some serotypes are associated with disease

outbreaks; large outbreaks of meningitis caused by serotype 1 have been reported from the African meningitis belt (9). The serotypes included in the currently licensed 7-valent pneumococcal conjugate vaccine (PCV7) were selected in part on the distribution of serotypes associated with invasive disease among U.S. children. Before the vaccine was introduced into the United States in 2000, the serotypes included in the vaccine caused approximately 80% of invasive infections in children younger than five years (10). On the basis of data available in 2000, the proportion of infections caused by 7valent serotypes is more than 50% in all regions of the world but with regional variation. Nevertheless, it appears that in some parts of Africa, Asia, and Europe, serotypes 1 and/or 5 account for a significant proportion of invasive infections in children, and thus are important for future vaccines (8). Most pneumococcal infections can be treated effectively with antibiotics, although meningitis often results in poor outcomes even with therapy. Antimicrobial resistance among S. pneumoniae complicates treatment. Pneumococci that are resistant to penicillin, erythromycin, co-trimoxazole, or multiple drugs are common in many regions (11). To date, antimicrobial resistance has been found in a relatively small number of serotypes; of 43 global resistant clones listed by the Pneumococcal Molecular Epidemiology Network as of 2006, about onehalf are PCV7 serotypes (12). Most pneumococcal infections result in transient, asymptomatic nasopharyngeal carriage, and only a small fraction go on to serious illness. Disease occurs when pneumococci leave the nasopharynx and enter the paranasal sinuses, middle ear, lungs, or blood stream. Carriage is more common and prolonged among children than among adults. All or nearly all children carry one or more serotypes before reaching age two years, and 50% or more acquire their first serotype in the first six months of life (13). Carriage is generally more common and occurs earlier among children in developing countries than those in industrialized settings. In developing country populations, carriage rates reach 30% to 50% as early as 6 to 14 weeks of age, whereas in the United States, the prevalence in children aged three to nine months is generally approximately 25% (13). The distribution of serotypes found in carriage studies from developing countries is generally more diverse than that seen in studies of invasive disease (14).

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PNEUMOCOCCAL VACCINES Most pneumococcal strains causing severe disease are surrounded by a characteristic polysaccharide capsule, a major virulence factor that allows the bacteria to evade phagocytosis. Pneumococcal vaccines containing purified polysaccharides from a subset of serotypes have been available for over 60 years. The 23-valent pneumococcal polysaccharide vaccine (Pneumovax1 23, Merck and Company, Inc., Whitehouse Station, New Jersey, U.S.), generally used in older children and adults with high-risk medical conditions and adults
65 years, has been available since 1983. Vaccines comprising polysaccharide antigens alone, however, produce weak or short-lived immune responses in infants and toddlers (15). Covalently linking pneumococcal polysaccharide to a carrier protein induces a T-cell-dependent immune response that can occur even in early infancy. A PCV was licensed for use in infants and young children in the United States in 2000. The vaccine [known as Prevnar1 (Wyeth, Madison, New Jersey, U.S.) in the United States and Prevenar1 elsewhere] includes capsular saccharides of seven serotypes (4, 6B, 9V, 14, 18C, 19F, 23F), each coupled to a nontoxic variant of diphtheria toxin, CRM197. As of 2008, the PCV7 remains the only formulation that is currently licensed, although two other formulations with 10 and 13 serotypes, respectively, are in phase III clinical trials and may be available between 2008 and 2010 (Table 1). Other pneumococcal conjugate formulations have also undergone phase III testing; for example, two clinical trials of a PCV9 manufactured by Wyeth showed significant protective efficacy in African infants (16,17), but the formulation was not ultimately licensed. The pneumococcal vaccine pipeline contains at least 20 different vaccine candidates in various stages of testing, including projects by emerging market manufacturers and projects to develop alternatives to PCVs (e.g., common protein vaccines). These vaccines are in early stages of development and will likely become available between 2015 and 2020.

CLINICAL TRIALS OF PNEUMOCOCCAL CONJUGATE VACCINES Carriage Studies evaluating PCV effects on carriage suggest that vaccination reduces acquisition of vaccine-type strains (13). In most but not all studies, carriage of nonvaccine-type strains increased among children receiving conjugate vaccine so that the overall prevalence of pneumococcal carriage was not different in vaccinated and unvaccinated children. Among toddlers in Israel, vaccination reduced carriage of antibioticresistant strains in vaccinated children and in their unvaccinated siblings through reduced transmission (18).

Otitis Media Clinical trials also have evaluated the effects of conjugate vaccines on otitis media. In a large randomized, double-blinded

clinical trial in Northern California, infants receiving PCV7 had 7% fewer episodes of otitis media, 9% fewer infants with frequent otitis media, and 20% fewer children requiring ventilatory tube placement compared with controls (19). In Finland, infants receiving PCV7 had 6% fewer episodes of otitis media overall and 57% fewer episodes of otitis media caused by pneumococci of vaccine serotypes (20). Notably, children in the pneumococcal vaccine group had 33% more episodes of otitis media caused by serotypes not in the vaccine or related to vaccine types. In Israel, a trial of a PCV9 evaluated otitis media and other upper respiratory tract infections among children of 12 to 35 months attending day care centers (21). Vaccination reduced episodes of otitis media but the change was not statistically significant (17% fewer episodes, 95% CI 2% to 22%). Significant reductions were seen for upper and lower respiratory tract infections and days of antibiotic use. Conversely, in the Netherlands, in children aged 1 to 7 years with a history of recurrent otitis media, PCV7 in combination with 23-valent pneumococcal polysaccharide vaccine showed no significant benefit for reducing ear infections (22). More recently, a randomized, controlled-trial of an 11-valent pneumococcal vaccine formulation from GlaxoSmithKline (precursor to the 10-valent formulation currently in development [Streptorix, Brentford, London, U.K.]) showed significant efficacy against culture-proven acute otitis media because of S. pneumoniae (23). This vaccine uses an outer membrane protein from Haemophilus influenzae as a carrier protein for the pneumococcal saccharides. Trial results indicated that the vaccine appeared to confer protection against otitis media because of nontypeable H. influenzae, in addition to otitis caused by vaccine-type pneumococci.

Invasive Disease and Pneumonia The efficacy of pneumococcal vaccines for prevention of invasive disease and pneumonia has been evaluated in five clinical trials. These trials include three different formulations from two different manufacturers (7- and 9-valent from Wyeth; 11-valent from Sanofi Pasteur, Lyon, France). In the Northern California Kaiser trial, vaccination reduced episodes of pneumonia confirmed by radiograph by 20% (Table 2) (24). A reanalysis of the trial using WHO criteria for pneumonia with consolidation on X ray found an efficacy of 30% (25). Efficacy against invasive disease caused by vaccine serotypes was 97% (19). In a second U.S. trial that employed community randomization, the 7-valent conjugate vaccine was found to be effective against invasive disease among Navajo and Apache children younger than two years, reducing episodes caused by vaccine serotypes by 83% (26). Two trials of PCV9 have been completed in developing countries. In South Africa, vaccination prevented invasive disease in both HIV-positive and HIV-negative infants, although point estimates of efficacy were higher in HIV-negative children

Table 1 Pneumococcal Conjugate Vaccines in Use or in Late-Stage Trials as of 2008 Manufacturer

Serotypes

Carrier protein

Stage

7-Valent conjugate (Prevnar, Prevenar) 10-valent conjugate (Streptorix)

Wyeth GSK

Diphtheria CRM197 Protein D

13-valent conjugate (Prevnar 13)

Wyeth

4, 6B, 9V, 14, 18C, 19F, 23F 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F 1, 3, 5, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F

Licensed, in use Submitted for licensure and in phase III trials Phase III trials

Diphtheria CRM197

Chapter 45: Pneumococcal Protein-Polysaccharide Conjugate Vaccines

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Table 2 Clinical Trials Measuring the Efficacy of Pneumococcal Conjugate Vaccine Against Invasive Pneumococcal Disease and Pneumonia in Young Children Reference

Population

Number of subjects

Pneumococcal vaccine

Black et al. (19,24); Hansen et al. (25)

Infants, California

37,868

7-Valent (Wyeth)

Klugman et al. (17)

O’Brien et al. (26)a

Infants, South Africa

Navajo and Apache <2 yr

Cutts et al. (16)

Infants, the Gambia

Lucero (27)

Children 3–24 mo, the Philippines

39,836

8,292

17,437

Outcome

Efficacy, % (95% CI)

Invasive disease, vaccine types Pneumonia, X-ray confirmed, clinical reading Pneumonia, X-ray confirmed, WHO-protocol readings Acute otitis media episodes

97 (83, 99) 20 (4.4, 34)

Pneumonia with alveolar consolidation, HIV Invasive disease, vaccine types, HIV Invasive disease, vaccine types, HIVþ Invasive disease, penicillin resistant

20 (2, 35)

Invasive disease, vaccine types, per protocol Invasive disease, vaccine types, intent-to-treat

77 (9.4, 95)

9-Valent (Wyeth)

Pneumonia, X-ray confirmed Invasive disease, vaccine types All-cause admissions Mortality

37 77 15 16

11-Valent (Aventis)

Pneumonia, X-ray confirmed

23 (1.1, 41)

9-Valent (Wyeth)

7-Valent (Wyeth)

30 (11, 46) 7.0 (4.1, 9.7)

83 (39, 97) 65 (24, 86) 67 (19, 88)

83 (21, 96) (27, 45) (51, 90) (7, 21) (3, 28)

The results listed are from per protocol analyses unless otherwise stated. a This clinical trial was community randomized; all others used individual randomization.

(Table 2) (17). Vaccination significantly reduced radiologically confirmed pneumonia in children who were HIV-negative. In the Gambia, PCV9 reduced radiologically confirmed pneumonia by 37% and invasive disease caused by vaccine serotypes by 77% (16). The most striking findings in the Gambia trial were that vaccination reduced hospital admissions and deaths from any cause by 15% and 16%, respectively. In the Philippines, an 11-valent conjugate vaccine made by Aventis (Sanofi Pasteur) reduced X-ray confirmed pneumonia among children of 3 to 24 months by 22.9% (95% CI, 1.1% to 41.2%). This point estimate is consistent with results from other trials but did not reach statistical significance on the basis of the small number of events observed. The overall efficacy masks significant heterogeneity in age-specific efficacy. Among children of 3 to 11 months, the efficacy was 34% (95% CI, 4.8– 54.3%), while among children of 12 to 23 months, the efficacy was 2.7% (95% CI, 43.5 to 34.0%) (27).

serotypes persists for at least three years. Duration of protection was also evaluated among participants of the South Africa trial, which used a three-dose infant schedule without a booster and evaluated PCV9 (29). The study reevaluated immunogenicity a mean of 5.6 years after vaccination and continued blinded surveillance for invasive pneumococcal disease for a mean follow-up of 6.3 years after vaccination. Among HIV-uninfected children, antibody concentrations among vaccine recipients remained more often above protective levels compared with controls; vaccine efficacy remained significant against invasive pneumococcal disease (78%; 95% CI 34–92%) and was similar to that found at a mean follow-up of 2.3 years (83%). In contrast, HIV-infected children showed evidence of waning immunity with anti-capsular antibody levels below 0.35 mg/mL and not significantly different from controls for three of seven serotypes evaluated; vaccine efficacy for this group fell from 65% at 2.3 years to 39% (95% CI 7.8 to 65%).

Duration of Vaccine Protection

VACCINE SAFETY

Existing data suggest that PCV7 may provide long-lasting immunologic memory and protection as other conjugate vaccines have done. The best data on duration of protection may come from longer-term follow-up of subjects who participated in phase III clinical trials. Follow-up from the U.S. Native American trial, which used a four-dose schedule for infants, evaluated nasopharyngeal carriage three years following infant vaccination with PCV7 (28). This study found that carriage of serotypes included in the vaccine remained significantly less common among vaccine recipients than among controls, suggesting that protection against acquisition of vaccine-included

Data from phase III clinical trials and smaller phases I and II studies suggest that PCVs are generally well tolerated. Typically, rates of mild, self-limited, local reactions such as redness, swelling, and tenderness were within the range of what is seen for other routinely received vaccines. Fever rates in the U.S. Kaiser trial were higher among children receiving PCV7 than among controls (19) and reported fever, but not measured fever, was higher among PCV9 recipients in the Gambia trial (16). In the Gambia, significantly more outpatient visits were made within a week after dose 1 among those receiving PCV9, but this difference was not seen with later doses (16).

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The rate of deaths and hospitalizations was not higher among those receiving PCV7 in the Kaiser trial (19). In the Gambia trial, the number of hospital admissions and deaths within seven days of receiving any dose of vaccine was similar in the group receiving PCV9 compared with the control group (16). In South Africa, rates of viral pneumonias requiring hospitalization within the first week after vaccination and asthma-related diagnoses at any time following vaccination were somewhat higher among PCV9 recipients (17). Post-marketing studies of the safety of PCV7 suggest that the vaccine is as safe as other routinely used vaccines. According to a summary of data from the Vaccine Adverse Event Reporting System (VAERS), a collection of passive reports of adverse events that are possibly related to vaccinations given in the United States, the majority of reports in children younger than 18 years of age during the first two years after licensure described minor adverse events similar to those previously identified in clinical trials (30). During this time, approximately 31 million doses were distributed and VAERS received 4154 reports of events that had occurred within three months of receiving PCV. Serious events were described in 14.6% of reports, a proportion consistent with the proportion of serious adverse events (14.2%) reported from VAERS for other vaccines.

ROUTINE USE AND EFFECTS OF PNEUMOCOCCAL CONJUGATE VACCINE As of early 2008, PCV7 has been licensed for routine use in more than 70 countries, and 17 (all high-income) countries have national immunization programs that provide access to PCV7 for all children (Fig. 1). In 2008, several middle-income countries will begin joining the high-income countries with routine immunization programs funded by their own national resources,

while at least two of the world’s poorest countries are expected to also begin vaccinating with support from the Global Alliance for Vaccines and Immunization (GAVI) Alliance.

Vaccine Effectiveness Introduction of PCV in 2000 led to a rapid drop in rates of invasive pneumococcal disease (i.e., bacteremia, bacteremic pneumonia, and meningitis), pneumonia, and otitis media in U.S. children. According to the Centers for Disease Control and Prevention’s (CDC’s) Active Bacterial Core surveillance (ABCs), a population-based system measuring invasive pneumococcal disease in approximately 20 million people in eight states, incidence of invasive disease started dropping in children younger than five years late in the first year the vaccine was licensed (Fig. 2); total incidence of invasive pneumococcal disease among children younger than five years dropped 75% from 99 cases per 100,000 population during 1998 and 1999 to 23 cases per 100,000 population in 2005; disease caused by vaccine-type strains fell 98% from 82 cases per 100,000 population to 1.7 (32,33). Incidence of disease caused by antibioticresistant strains also fell (34). At the same time, a relatively small but statistically significant increase was seen in invasive disease caused by some nonvaccine-type strains (so-called serotype replacement), in particular serotype 19A (35). A multicenter U.S. study of children requiring hospitalization for invasive pneumococcal disease reported similar findings to that seen in the ABCs areas (36) as have most surveillance programs evaluating invasive pneumococcal disease in single geographic areas in the United States (37–41). Among children who received medical care from Intermountain Health Care in Utah, however, the overall reduction in invasive disease was smaller than seen elsewhere, with only a 27% reduction in total disease rates by 2003 (42).

Figure 1 National vaccine programs using pneumococcal conjugate vaccine in routine schedules as of January, 2007. Source: From Ref. 31.

Chapter 45: Pneumococcal Protein-Polysaccharide Conjugate Vaccines

Figure 2 Incidence of invasive pneumococcal disease by year and year of age for children younger than five years in the United States. Source: From Ref. 33.

Use of conjugate vaccine in children has also reduced the differences in risk of invasive pneumococcal disease among certain racial and ethnic groups. According to ABCs data, incidence among black children younger than two years went from 3.3 times the rate among white children in the pre-vaccine period to 1.6 times the rate among white children in 2002 (43). In Alaska, vaccine-type invasive disease fell 91% among Alaska Native children younger than two years and 80% among nonNatives the same age after vaccine introduction, eliminating the disparity in disease caused by vaccine serotypes (5) More recent data indicate, however, that an increase in invasive disease caused by nonvaccine-type pneumococci (primarily serotype 19A) may be eroding the benefit that PCV7 is showing among Alaska Natives (44). Observational studies also show that PCV7 use is associated with declines in noninvasive syndromes caused in part by pneumococcal infection. Studies using administrative data sets from the United States have found significantly lower rates of otitis media, pressure equalizing tube placement, recurrent otitis media, and pneumonia in children following conjugate vaccine introduction compared with what was expected from pre-vaccine rates (45–47). Analysis of a nationwide sample found that rates of pneumonia hospitalizations among children younger than two years dropped 39% (506 cases/100,000 children) comparing 2004 to the years before vaccine licensure; the decrease represents approximately 41,000 fewer pneumonia hospitalizations in the United States in 2004 (46). In Italy, a cohort study of impact of vaccination at 3, 5, and 12 months showed less X-ray confirmed pneumonia, acute otitis media, and antibiotic use in children who had received PCV7 compared with those that had not; the effects were statistically significant after age 12 months (48). Published surveillance data on vaccine impact from outside the United States are currently limited. In Canada, the provinces of Alberta and Nunavut were the first jurisdictions to implement routine PCV programs, in September 2002. The effect on vaccine burden was similar to if not more rapid than that seen in the United States When compared with the

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combined rate between 1998 and 2001, the rate among children aging 23 months and younger decreased by 82% to 12 cases per 100,000 in 2004 for all serotypes and by 93% to 4 cases per 100,000 for vaccine serotypes (49). In Australia, early data suggested a reduction in cases of PCV7-serotype disease in indigenous children younger than two years (6). Epidemiological studies have also been used to evaluate effectiveness of PCV7. Two surveillance programs within the United States—the U.S. Pediatric Multicenter Pneumococcal Surveillance Group and the Massachusetts Department of Public Health—combined their data and used a case-only method to estimate the effectiveness of abbreviated or delayed dosing regimens against invasive pneumococcal disease (50). In children not at high risk for invasive disease, the effectiveness of the vaccine against vaccine serotypes was estimated to be 91% for the full four-dose schedule, adjusting for study year (Table 3). Effectiveness was somewhat higher when measured in a large case-control study that used cases of invasive disease identified through CDC’s ABCs multisite surveillance program and age-matched controls. This study found that one or more doses of conjugate vaccine was 96% effective against invasive disease in healthy children, 81% effective in children with comorbid medical conditions (Table 3), and 76% effective overall against disease caused by strains resistant to penicillin (51). Vaccination was shown to be significantly protective against all seven individual vaccine serotypes and vaccinerelated serotype 6A, but not against vaccine-related serotype 19A. In Spain, a case-control study found similarly good protection against invasive disease caused by vaccine serotypes, but also noted an increase in risk of nonvaccine-type disease among vaccine recipients, a finding that was not seen in the other studies and remains unexplained (52).

Vaccine Schedules Clinical trials of PCV7 conducted in the United States used a four-dose series, with doses given at ages 2, 4, 6, and 12 months (19,26); therefore, PCV7 was first licensed as a four-dose series. Schedules for routine infant immunizations in many other parts of the world use a three-dose series, however. In general, the four-dose infant schedules recommend three doses at defined time periods within the first six months of age and a fourth dose at or after one year. Three-dose schedules are either three doses in the first six months without a booster or two doses in the first six months followed by a third dose around age one year. A growing body of evidence suggests that using fewer than four infant doses is protective. Phase III trials of the 9valent vaccine in South Africa and the Gambia showed that a three-dose primary series conferred substantial protection (16,17). In the Northern California Kaiser Permanente trial, efficacy for partially vaccinated children was 85.7% (95% CI 0%, 100) (19). In Italy, a recently published single-blind cohort study of vaccination at 3, 5, and 12 months showed less X-ray confirmed pneumonia, acute otitis media, and antibiotic use in children who had received PCV7 compared with those that had not, but the effects were statistically significant after age 12 months (48). Immunogenicity studies also support the potential for fewer than four doses to provide substantial protection against invasive disease (53). A large case-control study evaluating effectiveness against invasive disease in the United States provided estimates of vaccine effectiveness for multiple partial and complete schedules and found that nearly all schedules provided

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Table 3 Observational Studies Evaluating Effectiveness of Pneumococcal Conjugate Vaccine Against Invasive Disease Caused by Vaccine Serotypes in Young Children Population

Study design

Number subjects

Schedule

U.S. children 3–59 mo, multisite (51)

Case control

3294 (782 cases, 2512 controls)


1 dose, any age 1 dose 7 mo 2 doses 7 mo 3 doses 7 mo 3 doses 7 mo and 1 dose 12–16 moa 1 dose 12–23 mo 2 doses 12–23 moa 1 doses
24 moa

Healthy children <5 yr identified in Massachusetts and through 8 U.S. pediatric hospitals (50)

Children <5 yr, Navarra, Spain (52)

Case only (indirect cohort)

Case control

400

510 (85 cases, 425 controls)

Vaccine effectiveness, % (95% CI) Healthy: 96 (93, 98) Comorbid conditions: 81 (57, 92) 73 (43, 87) 96 (88, 98) 95 (88, 98) 100 (94, 100) 93 (68, 98) 96 (68, 98) 94 (49, 99)

1 dose <3 mo 2 doses <5 mo 3 doses <7 mo 3 doses <7 mo and 1 dose 12–15 moa 1 dose 12–23 mo 2 doses 12–23 moa

39 70 77 91


1 dose, any age Incomplete Complete (1–3 doses depending on age)

88 (9, 98) 100 (N/A) 81 (54, 97)

(80, 80) (28, 88) (50, 89) (18, 99)

55 (241, 94) 68 (219, 97)

a

Fully vaccinated according to the recommended U.S. schedule.

some protection compared with no vaccine, although a single dose at less than seven months was less protective than two or three doses received at less than seven months (Table 3). A direct comparison of three doses before seven months plus a booster at 12 to 15 months with three doses before seven months without a booster suggested that the booster added additional protection (p ¼ 0.03) (51). A second study using combined data from two surveillance programs within the United States and a case-only method also showed additional protection from the fourth dose compared with three doses only, but the point estimates of protection (50). The benefit of a booster dose for children in developing countries is less clear, given the high magnitude of antibodies obtained after the three-dose primary series and the duration of protection noted among HIV-uninfected South African children in spite of a lack of booster dose (29).

Impact on Carriage and Herd Immunity An important component of the success of PCV has been its ability to reduce acquisition of carriage in vaccinated children, thereby reducing transmission of vaccine-type pneumococci and preventing disease in unvaccinated children and adults (socalled herd or indirect effects). In Massachusetts, a crosssectional study of nasopharyngeal carriage among children younger than seven years visiting primary care practices found that vaccine-serotype pneumococci accounted for only 14% of colonizing strains in 2004, a decrease from 36% in 2001; at the same time, nonvaccine serotypes increased so that overall carriage did not change substantially (from 26% to 23% of all children) (54). In Alaska, a series of studies have evaluated carriage among Alaska Natives living in rural villages and in urban settings (5,55). In both settings, vaccine-type colonization

decreased over time (from 55% of pneumococci at baseline to 11% in 2003 among residents of villages <5 years), while carriage of nonvaccine-type pneumococci increased; vaccinetype carriage was significantly more common among children who were incompletely vaccinated or unvaccinated. Carriage of vaccine-type pneumococci also fell among adults following vaccination of children (55). In the United States, invasive disease in adults 65 years and older has dropped by about one-third since introduction of PCV for children, according to data from CDC’s ABC surveillance (Fig. 3). The change has been caused by an approximately 80% reduction in disease caused by PCV7 serotypes between 1999 and 2005; a small increase has been noted in disease caused by other pneumococcal serotypes, including those contained in the 23-valent pneumococcal polysaccharide vaccine but not in PCV7 (35). A drop of similar magnitude was seen in hospitalizations for pneumococcal bacteremia in older adults as measured using a large database from Medicare, the U.S. government’s system for providing health care for the elderly (56). Some, but not all, other high-risk groups are indirectly benefiting from PCV7 use in infants and young children. Following PCV7 introduction, invasive disease caused by PCV7 serotypes fell by about half among newborns and infants too young to have been vaccinated (57). Between pre-vaccine years (1998 and 1999) and 2003, an overall reduction of 19% in invasive disease rates was noted among adults of 18 to 64 years with HIV or AIDS, a group with disease rates up to 100 times that of healthy adults of the same age (58). Of note, the overall figure represented a 62% drop in disease caused by vaccine serotypes and a concomitant 44% increase in disease caused by nonvaccine serotypes; no increase was seen in nonvaccine-type disease in adults of 18 to 64 year who did

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among young children each year globally. The clinical trials and studies of impact of PCV introduction have led to a greater understanding of pneumococcal disease, in particular the differences among serotypes and patterns of transmission. Some of the remaining questions include how the new vaccine formulations will work in practice in a variety of settings, the number and timing of doses needed to induce long-term protection, and whether the emergence of nonvaccine serotypes will create a need to modify conjugate vaccine formulations over time.

REFERENCES

Figure 3 Rates of invasive pneumococcal disease among adults
65 years by serotype and year. Source: From Ref. 35.

not have HIV or AIDS, although this group also had a reduction in PCV7-type disease of over 60%. A reduction in disease caused by PCV7 serotypes has been noted among Alaska Native adults, but because PCV7 serotypes accounted for a small proportion of cases and invasive disease caused by other serotypes has increased somewhat, invasive disease rates in Alaska Native adults have not changed following PCV7 introduction (5). While use of PCV7 in other settings would be expected to similarly reduce transmission of vaccine-type strains, the size of the overall benefit from the indirect effects will depend on the amount of disease caused by vaccine serotypes.

LOOKING FORWARD In summary, PCVs have shown remarkable efficacy in clinical trials and one formulation, PCV7, has now shown major benefits in the United States and other industrialized countries in routine use. PCV7 has reduced invasive disease, pneumonia, and otitis media caused by vaccine types as well as disease caused by antibiotic-resistant pneumococci not only in children who are vaccinated but also in older persons and infants too young to have received vaccine. An increase in nonvaccinetype disease, primarily caused by serotype 19A, has been noted, but to date the magnitude of this increase has been small compared with the decreases seen in vaccine-type disease. On the basis of these findings, an increasing number of countries globally are introducing PCV. New conjugate formulations containing antigens targeting 10 and 13 serotypes are in late stages of development. The vaccines will contain serotypes 1 and 5, serotypes that will improve their usefulness in developing-country settings. Looking forward, a remaining challenge is to ensure that uptake of these vaccines occurs as rapidly as possible into the populations that need them most, as their licensure and distribution is likely to hundreds of thousands of deaths

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35. Hicks LA, Harrison LH, Flannery B, et al. Incidence of pneumococcal disease due to non-pneumococcal conjugate vaccine (PCV7) serotypes in the United States during the era of widespread PCV7 vaccination, 1998–2004. J Infect Dis 2007; 196: 1346–1354. 36. Kaplan SL, Mason EOJ, Wald ER, et al. Decrease of invasive pneumococcal infections in children among 8 children’s hospitals in the United States after the introduction of the 7-valent pneumococcal conjugate vaccine. Pediatrics 2004; 113:443–449. 37. Haddy RI, Perry K, Chacko CE, et al. Comparison of incidence of invasive Streptococcus pneumoniae disease among children before and after introduction of conjugated pneumococcal vaccine. Pediatr Infect Dis J 2005; 24:320–323. 38. Hsu K, Pelton S, Karumuri S, et al. Massachusetts Department of Public Health Epidemiologists. Population-based surveillance for childhood invasive pneumococcal disease in the era of conjugate vaccine. Pediatr Infect Dis J 2005; 24:17–23. 39. Shafinoori S, Ginocchio CC, Greenberg AJ, et al. Impact of pneumococcal conjugate vaccine and the severity of winter influenza-like illnesses on invasive pneumococcal infections in children and adults. Pediatr Infect Dis J 2005; 24:10–16. 40. Mufson MA, Stanek RJ. Epidemiology of invasive Streptococcus pneumoniae infections and vaccine implications among children in a West Virginia community, 1978–2003. Pediatr Infect Dis J 2004; 23:779–781. 41. Black S, Shinefield H, Baxter R, et al. Impact of the use of heptavalent pneumococcal conjugate vaccine on disease epidemiology in children and adults. Vaccine 2006; 24(suppl 2):S2–79–80. 42. Byington CL, Samore MH, Stoddard GJ, et al. Temporal trends of invasive disease due to Streptococcus pneumoniae among children in the intermountain west: emergence of nonvaccine serogroups. Clin Infect Dis 2005; 41:21–29. 43. Flannery B, Schrag S, Bennett NM, et al. Impact of childhood vaccination on racial disparities in invasive Streptococcus pneumoniae infections. JAMA 2004; 291:2197–2203. 44. Hennessy T, Singleton RJ, Bulkow LR, et al. Increase in invasive pneumococcal disease in Alaska Native children due to serotypes not in the heptavalent pneumococcal conjugate vaccine, 2001– 2005. 5th International Symposium on Pneumococci and Pneumococcal Diseases, Alice Springs, Australia 2006; Abstract No. SY2.06:60. 45. Poehling KA, Lafleur BJ, Szilagyi PG, et al. Population-based impact of pneumococcal conjugate vaccine in young children. Pediatrics 2004; 114:755–761. 46. Grijalva CG, Nuorti JP, Arbogast PG, et al. Decline in pneumonia hospitalizations following routine childhood immunization with pneumococcal conjugate vaccine in the United States. Lancet 2007; 369:1179–1186. 47. Poehling KA, Szilagyi PG, Grijalva CG, et al. Reduction of frequent otitis media and pressure-equalizing tube insertions in children after introduction of pneumococcal conjugate vaccine. Pediatrics 2007; 119:707–715. 48. Esposito S, Lizioli A, Lastrico A, et al. Impact on respiratory tract infections of heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 11 months of age. Respir Res 2007; 8:12. 49. Kellner JD, Church DL, MacDonald J, et al. Progress in the prevention of pneumococcal infection. CMAJ 2005; 173:1149–1151. 50. Mahon BE, Hsu K, Karumuri S, et al. Effectiveness of abbreviated and delayed 7-valent pneumococcal conjugate vaccine dosing regimens. Vaccine 2006; 24:2514–2520. 51. Whitney CG, Pilishvili T, Farley MM, et al. Effectiveness of sevenvalent pneumococcal conjugate vaccine against invasive pneumococcal disease: a matched case-control study. Lancet 2006; 368:1495–1502. 52. Barricarte A, Castilla J, Gil-Setas A, et al. Effectiveness of the 7-valent pneumococcal conjugate vaccine: a population-based case-control study. Clin Infect Dis 2007; 44:1436–1441. 53. Goldblatt D, Southern J, Ashton L, et al. Immunogenicity and boosting after a reduced number of doses of a pneumococcal

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46 Pneumococcal Common Proteins and Other Vaccine Strategies David E. Briles Departments of Microbiology and Pediatrics, The University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.

James C. Paton School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia

Susan K. Hollingshead Department of Microbiology, The University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.

John W. Boslego Vaccine Development Global Program, PATH, Washington, D.C., U.S.A.

DEVELOPMENT OF NOVEL VACCINES TO PNEUMOCOCCAL ANTIGENS Streptococcus pneumoniae expresses a number of molecules aside from capsular polysaccharide that are able to elicit protection (Fig. 1). This chapter summarizes data for some of the molecules for which the most complete data have been obtained in animal studies (Table 1). Space constraints prevent description of all potential antigens and all relevant citations.

PHOSPHOCHOLINE/TEICHOIC ACIDS Phosphocholine (PC) is a common epitope on several respiratory bacterial pathogens (1). PC is an invariant epitope of the teichoic and lipoteichoic acids of all pneumococci, and the demonstrations that mouse and human (2,3) antibodies to PC can protect mice from fatal infection provided the first evidence that a defined antigen other than capsular polysaccharides could elicit protection against pneumococci. However, the PC-epitope on teichoic and lipoteichoic acids does not elicit memory responses; moreover, antibodies to PC are less protective per molecule than those to capsule (4), and isolated PC-containing teichoic acid is not highly immunogenic.

PNEUMOCOCCAL PROTEIN VACCINE ANTIGENS Protection-eliciting pneumococcal protein(s) provide an attractive alternative to using capsular polysaccharides or polysaccharideprotein conjugates as a vaccine (5). Infants generally make good responses to protein antigens, and the immunogens in successful nonliving pediatric vaccines are generally proteins themselves, or are associated with protein carriers. It should be possible to target several critical virulence and invasion mechanisms by using more than one protein in a vaccine. Immunization with mixtures of pneumococcal proteins can be more protective in mice than immunization with individual proteins (6–8). Since the recombinant proteins are relatively inexpensive to produce, once the substantial costs of performance of clinical

trials and the construction of a manufacturing facility have been resolved, a protein vaccine could be affordable worldwide. The first pneumococcal proteins shown to elicit protection in mice were pneumolysin, PspA, neuraminidase, autolysin, PspC, and PsaA. Each of these proteins was found to play a role in virulence (9). Based on analysis of the pneumococcal genome, the use of in vivo gene selection systems, and identification of antigens recognized by human sera, many additional potential protein vaccine candidates have been identified. Many of these are described below.

PspA PspA (pneumococcal surface protein A) is expressed by virtually all pneumococci (10) and has been used to immunize human volunteers (11). Human antibodies elicited by immunization with rPspA protect mice from fatal sepsis with pneumococci (12). PspA molecules range in size from about 65 kDa to about 95 kDa (13). The N-terminal a-helical half of the molecule has an antiparallel coiled coil structure (14), is surface exposed (15), and is protection eliciting (12). The center of the molecule contains about 80 residues, 40% of which are proline. Over half of PspA molecules have a 33-residue highly conserved nonproline-containing region in the center of the proline-rich region. The proline-rich region and its nonproline block are surface-exposed and protection eliciting. The C-terminal end of the molecule contains conserved choline-binding repeats that attach PspA to the PC residues of the cell surface lipoteichoic acids. Although PspA exhibits structural variability (13), protective antibodies elicited to the N-terminal coiled coil domain and proline-rich region are quite cross-reactive (12,16). Ninetyeight percent of PspAs exist in two cross-protective PspA families (10,13,17). Antibodies elicited to PspA in mice, rabbits, and humans can protect mice from intravenous infection with pneumococcal challenge strains (9). It is expected that a PspA vaccine will not need more than three PspAs (12,18).

Chapter 46: Pneumococcal Common Proteins and Other Vaccine Strategies Table 1 Noncapsular Pneumococcal Antigens That Have Exhibited Protective Efficacy in Mice Protective against Noncapsular antigen Autolysin NanA PcpA PcsB Phosphocholine/teichoic acids PhtB PhtE PiaA Pillin PiuA Pneumolysin PotD PsaA PspA PspC StkB StkP Whole-killed-pneumococci

Invasive disease þ þ þ þ þ þ þ þ þ þ þ þ – þ þ þ þ þ

Nasal colonization þ  ?

 þ þ þ þ þ

þ, indicates data strongly supporting protective effects; , indicates convincing evidence for an absent or only weak effect; ?, positive report that needs confirmation.

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Protection against pneumococcal infection is highly dependent on the ability of complement (C0 ) to opsonize pneumococci. In the absence of antibody, C0 deposition occurs through the classical pathway (19). PspA blocks C0 deposition triggered through the classical pathway by blocking deposition of C1q to the pneumococcal surface (20,21). Antibody to PspA enhances C0 deposition (21). Most of the C’ deposited after triggering of the classical pathway is dependent on the amplification loop of the alternative pathway. PspC (also called CbpA) binds factor H and as a result inhibits the alternative pathway (22). Thus, pneumococci lacking PspA and PspC exhibit much higher levels of C0 deposition than pneumococci lacking either one (20,23). All PspA families and clades bind lactoferrin avidly and specifically (24,25). The binding site for lactoferrin is within the a-helical domain (14,25). The ability of PspA to bind to lactoferrin allows PspA to protect pneumococci from attack by apolactoferrin and lactoferrin (26). Apolactoferrin is in all body secretions at concentrations from 0.1 to 7 mg/mL, with the highest concentrations being associated with inflammation (27). Antibody to PspA prevents PspA from binding lactoferrin and increases killing by apolactoferrin. Antibody to PspA is initially acquired transplacentally from the mother (28). Acquisition by children of their own antiPspA antibodies occurs during the period of time in which young children begin exhibiting relative resistance to pneumococcal infection (29–32).

Pneumolysin

Figure 1 Cartoon of many of the protection-eliciting molecules of the pneumococcus. The cell membrane and cell wall (nonbold type) have not been shown to elicit protection, although molecules attached to them can elicit protection. Antigenic portions of capsular polysaccharides, teichoic acids, and lipoteichoic acids are polysaccharides. Phosphocholine is a protection-eliciting epitope of teichoic and lipoteichoic acids. Pneumolysin is a cytoplasmic protein released during growth and autolysis. The remaining molecules are all proteins. Several of the proteins have choline binding domains that allow them to attach to lipoteichoic and teichoic acids.

Pneumolysin is produced by virtually all strains of S. pneumoniae, and was one of the first pneumococcal proteins proposed as a vaccine antigen (33). It is a member of the family of thiolactivated cytolysins. These toxins initially interact with cholesterol in host cell membranes and then insert into the bilayer and oligomerize to form transmembrane pores, thereby bringing about cell lysis (34). The ubiquity of cholesterol in animal cell types accounts for the broad range of effects attributable to these toxins, many of which occur at sub-lytic concentrations. These effects include inhibition of the bactericidal activity of leukocytes, blockage of lymphocyte proliferation and Ig production, reduction of ciliary beating of human respiratory epithelium, and direct cytotoxicity for endothelial and epithelial cells (35). Pneumolysin also binds the Fc region of human IgG and activates the classical complement pathway (reducing serum opsonic activity) (36,37). Thus, pneumolysin can (i) interfere with phagocytic and ciliary clearance of pneumococci, (ii) block humoral immune responses, and (iii) aid penetration of host tissues. Pneumolysin also induces inflammatory responses (38) and can reduce their ability to protect against infection (39). Injection of pneumolysin into rat lungs induces lobar pneumonia, indistinguishable histologically from that caused by virulent pneumococci (40). Pneumolysin is necessary for full virulence in mouse models of sepsis and pneumonia (41) and pneumolysin mutants exhibit reduced injury to the alveolar-capillary barrier and show a delayed onset of bacteremia (42,43). Pneumolysin is a highly conserved protein. Extensive analysis of pln genes has revealed negligible variation in its deduced amino acid sequence. The toxicity of native intact pneumolysin makes it unsuitable as a human vaccine antigen. As a result, nontoxic but immunogenic ‘‘pneumolysoids’’ have been produced by introducing mutations in regions essential for its cytotoxic and/

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or complement activation properties (44). Immunization of mice with a pneumolysoid (PdB) carrying a Trp433-Phe mutation (resulting in >99.5% reduction in cytotoxicity) provided a significant degree of protection against all nine capsular serotypes of S. pneumoniae tested (45). Humans develop antibody to pneumolysin as a result of natural exposure to S. pneumoniae, and the elicited antibody can passively protect mice from pneumococcal disease (46). Thus, it is anticipated that pneumolysoids will elicit protection in humans. Antibodies to pneumolysin presumably impart protection by neutralizing the biological properties of the toxin rather than by stimulating opsonophagocytic clearance of the invading bacteria.

PspC PspC (also called CbpA, SpSA, or Hic) is another pneumococcal surface protein (47–50). This virulence protein plays a role in adherence and colonization (47,51), and immunity to PspC can protect against colonization and invasive disease (49,51,52). PspC binds secretory IgA (48), factor H binding (50,53,54), and the polyimmunoglobulin receptor of the host (55). The binding of PspC to factor H results in inhibition of alternative pathway C3 activation (22). The multiple names for PspC came from the different activities of the protein and because of its variable mosaic protein, which includes alleles encoding very different domain structure (49,50,56). PspC was the first name given to this family of alleles in Gene Bank (09/26/1996). The domain structure of many PspC molecules is reminiscent to that of PspA. The range of sizes of PspA and PspC overlap, and some PspC molecules have a-helical domains that are highly homologous to portions of the a-helical domain of some PspA molecules (49). The proline-rich domains of PspC are very similar to and frequently indistinguishable from those of PspA. The PspC of about 75% of pneumococci have a choline-binding domain indistinguishable from PspA (49,57). The remaining pneumococci produce a PspC called Hic in which the choline-binding region is replaced by an LPXTG motif associated with attachment of proteins to the cell wall peptide cross-bridge by the enzyme sortase (50,56). Eleven major groups of PspC proteins exist, and a nomenclature based on differences in the domain structures of the encoded proteins has been proposed (56).

Pneumococcal Surface Antigen A PsaA is the metal-binding lipoprotein component of an ATPbinding cassette (ABC) transport system with specificity for Mn2þ (58). Defined PsaA mutants of S. pneumoniae are virtually avirulent for mice and exhibit markedly reduced adherence in vitro to human type II pneumocytes (59). This is presumed to be a consequence of a requirement for Mn2þ as a cofactor or for regulation of expression of other virulence factors (e.g., adhesins), and/or growth retardation due to an inability to scavenge this metal in vivo (60,61). The avirulence of PsaA pneumococci might also be due to the fact that they are highly susceptible to oxidative stress (62). Although PsaA is less efficacious as a vaccine than pneumolysin or PspA, it confers partial protection against intraperitoneal challenge with S. pneumoniae (63). PsaA is only 7 nm at its longest axis (64), making it unlikely that when anchored to the outer face of the cell membrane via its N-terminal lipid moiety, it is well exposed on the outer surface of the pneumococcus. Thus, the observed protection against IP

challenge is presumably due to in vivo blockade of ion transport, resulting from antibody that has diffused through the capsule and cell wall. Intranasal (IN) immunization with PsaA leads to efficient protection against colonization (6), but this may be due to the effects of CD4 cells (65), which would not require surface exposure of PsaA.

Autolysin Autolysin, an N-acetylmuramoyl-L-alanine amidase, is a virulence factor (66,67) that is responsible for pneumococcal autolysis, following pneumococcal death. Isolated autolysin elicits protective immune responses (68). The observation that antibodies to autolysin and pneumolysin do not have synergistic protective effects suggests that the virulence mechanism of autolysin is the autolytic release of pneumolysin, which carries out the virulence functions (68). Mutations in either autolysin or pneumolysin in capsular type 2 strain D39 reduces virulence in a lung inoculation model (67).

Neuraminidase and Hyaluronidase Streptococcus pneumoniae produces a large number of hydrolytic enzymes, some of which degrade host glycoproteins or extracellular matrix (69). A few of these are known virulence factors, including the neuraminidases NanA and NanB and the hyaluronate lyase (70–72). NanA is capable of cleaving terminal sialic acid from host glycoconjugates and in so doing, unmasks targets for pneumococcal adhesins (73). Some NanA mutant strains are more efficiently cleared from the nose and lung (74,75). Immunization with NanA extends the life of mice in an IN sepsis model and also protects against colonization and otitis media in chinchillas (70,76). Hyaluronate lyase is predicted to be a surface-bound protein in the pneumococcus. It degrades hyaluronic acid, a component of basement membranes and connective tissue; a hyl mutant exhibited reduced virulence in a mouse bacteremia model (71).

PhtA, B, D, and E The histidine triad proteins, designated PhtA, B, D, and E, are potential vaccine candidates (77). Their function is unknown but they are surface-exposed, and antibodies to PhtA and PhtD are found in human convalescent sera. Some Pht proteins elicit immunity against systemic challenge with pneumococci (77,78). However, in a direct comparative study, the most promising of these (PhtB and PhtE) were found to be less efficacious than either pneumolysoid or PspA (8).

PiuA and PiaA The lipoproteins PiuA and PiaA are components of iron uptake transporters of S. pneumoniae. PiuA and PiaA are antigenically cross-reactive, and immunity to these proteins is reactive with pneumococci of nine different S. pneumoniae serotypes. Immunity to both PiuA and PiaA is more protective than immunity to either protein alone (79).

PotD PotD is the surface transporter molecule of an ABC transporter for polyamine, an important nutrient of pneumococci. PotD appears to be highly conserved and is essential for virulence at least in sepsis and pneumonia models. Active immunity and passive antibody to PotD is protective against sepsis, pneumonia, and colonization in mice (80).

Chapter 46: Pneumococcal Common Proteins and Other Vaccine Strategies

Pilus Subunit No more than about 20% pneumococci express pilin (81). For at least some of those strains, it is an important virulence factor and immunity to pilin is protective against weakly infectious doses of pneumococci making pillin (82). However, pneumococci lacking pillin can also be highly virulent. This finding illustrates how the great genetic diversity of pneumococci (69,81) has allowed them to develop multiple virulence genotypes, and emphasizes the importance of incorporating multiple proteins in any pneumococcal protein vaccine.

PcsB and StkP PcsB is a pneumococcal protein that appears to be homologous with a protein in group B streptococci that is required for cell wall separation, and StkP is a serine/threonine protein kinase. These two proteins are both highly conserved, and immunization with them individually elicits protection in mice against pneumococci of four different capsular types. Both proteins elicited protection similar to that of PspA against sepsis, but StkP provided better protection than PspA against focal lung infections (83).

PcpA PcpA is a leucine-rich protein shown to elicit protection against lung infection and sepsis, but not against colonization (84). Thus, along with pneumolysin, it might be able to protect against disease without inducing subsequent evolution of pneumococci to escape the vaccine.

COMBINATION PROTEIN VACCINES Virtually all the pneumococcal proteins under consideration as vaccine antigens are directly or indirectly involved in the pathogenesis of pneumococcal disease. Mutagenesis of some combinations of virulence factor genes, for example, those encoding pneumolysin and either PspA or PspC (71), or PspA and PspC (51), synergistically attenuate pneumococcal virulence in animal models, indicating that the respective proteins function independently in the pathogenic process. Such results suggest that immunization with combinations of these antigens might provide additive protection. Moreover, the individual antigens differ in their capacity to protect against different S. pneumoniae strains (45,85). Thus, a combined pneumococcal protein vaccine is expected to elicit a higher degree of protection against a wider variety of strains than any single antigen. To date, only a limited number of combination experiments have been performed. The combination of pneumolysoid PdB and PspA clearly provides enhanced protection against systemic infection and pneumonia (7,63). In a more recent study, combinations of PdB and PspA, PdB and PspC, and PspA and PspC were all more efficacious against systemic challenge than individual antigens, and the combination of all three antigens was best (8). The combination of PspA and PsaA provided additive protection against carriage (6). Thus, present data strongly support the importance of using more than one antigen in a protein-based pneumococcal vaccine. As additional antigens are examined, some may be found to be of significant value for use in new vaccine formulations.

PNEUMOCOCCAL PROTEINS AS CARRIERS FOR POLYSACCHARIDES Pneumococcal proteins could be used in combination with polysaccharide-protein conjugates or as carriers for conjugation

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of pneumococcal polysaccharides. By including cross-reactive protection eliciting proteins to a vaccine containing pneumococcal polysaccharides or polysaccharide-protein conjugates, the spectrum of protection elicited by the polysaccharide and conjugate vaccines could be broadened. Preparing pneumococcal polysaccharide conjugates using pneumococcal proteins rather than proteins of other species, such as diphtheria or tetanus toxoid, should also have an advantage in terms of anamnestic responses elicited by a pneumococcal infection. If the vaccine stimulates T-cell memory that can be boosted upon infection, a faster anamnestic antibody response may be achieved, than when the carrier in the vaccine is not presented by infecting pneumococci. Pneumolysin and PspA have both been successfully used as carriers for immune responses to polysaccharides in animals (44,86).

PROTECTION AGAINST NASAL COLONIZATION To achieve herd immunity against the pneumococcus, it is necessary to prevent pneumococcal colonization of the nasopharynx. Since invasion is invariably thought to be preceded by at least a short duration of carriage, protection against carriage should prevent subsequent invasion. PsaA, NanA, PspC, and PspA have all been shown to protect against colonization in mice when injected intranasally with an adjuvant such as cholera toxin B subunit or whole cholera toxin (6,51,87). Intramuscular immunization with appropriate pneumococcal proteins may also protect against carriage, since immunization with NanA by this route can reduce carriage in chinchillas (76). Mouse studies, however, indicate that mucosal immunization may be more effective at preventing carriage than systemic immunization (88), although the mechanism of protection following IN immunization is primarily by T cells rather than antibody (65). In an effort to develop a pneumococcal vaccine for the developing world, the use of killed nonencapsulated pneumococci for IN immunization has been investigated in mice and found to be efficacious (89). This approach requires no purification of antigen, conjugation of antigen or bacterial DNA, thus simplifying manufacture.

CLINICAL CONSIDERATIONS FOR A PNEUMOCOCCAL COMMON PROTEIN VACCINE Although laboratory investigations and preclinical evaluations of common pneumococcal proteins have been ongoing over several decades, only a few clinical trials have been conducted, and even fewer have been published (11,12). The published clinical trials evaluated the safety, tolerability, and immunogenicity of rPspA. Activity in this field has increased in the past five years, and a steady stream of clinical trials of various common proteins and combinations of proteins is expected. The two target populations for immunization with pneumococcal common protein vaccines are: (i) infants and young children and (ii) the elderly. For either population, a phase I clinical trial to evaluate safety and immunogenicity of the common protein vaccine in healthy adults will be conducted. For the infant and young children indication, a following study in toddlers will confirm the safety and immunogenicity. Stepping down to infants, a phase I/II dose-ranging study will investigate several vaccine dosages. The ultimate vaccine regimen for infants will likely consist of three to four injections administered at 6, 10, 14, and 52 weeks of age. The common protein vaccine will be administered concomitantly with

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standard infant vaccines (e.g., DTP) and evaluations will be needed to ensure that the new vaccine does not interfere with immune responses to the standard vaccines, especially the Haemophilus influenzae type b conjugate vaccine. Because the incidence of pneumococcal colonization in young infants is high, evaluation of the impact of the vaccine on acquisition of pneumococcal carriage could be readily assessed in early studies. While the absence of an impact on carriage should not halt development of a common protein vaccine, a moderate or large impact on carriage will constitute a highly encouraging benchmark and would suggest a high probability of vaccine effectiveness. During early-phase studies, it will be important to establish and validate serologic assays including functional assays to assess vaccine performance and to identify possible serologic correlates of protection that would be corroborated in phase III studies. Since the common protein vaccine may have a different mechanism of action from the capsular polysaccharide-based vaccines, a large phase III clinical end point study should be conducted. Since a phase III study is many years away, it is likely that pneumococcal conjugate vaccines (PCVs) will be in use in many locations. Thus, a PCV could become an active control, or a PCV could be administered to both treatment groups. In either case, the primary end point will be pneumococcal disease caused by serotypes not contained in the PCV. A secondary end point will be pneumococcal disease caused by all serotypes. Options for disease end points include radiologically confirmed pneumonia, clinical pneumonia, and invasive pneumococcal disease. It will be critical to develop sensitive and specific diagnostic tools such as PCR and/or antigen detection technologies to confirm that S. pneumoniae is the etiologic agent. Serogroup must also be determined since the primary analysis will be on cases of pneumococcal disease caused by serotypes not contained in the vaccine. The trial is likely to be large and complex. The sample size will depend upon the attack rates caused by serotypes not contained in the vaccine in the study area. Several years of effort will be needed to adequately prepare to conduct the study. Clinical development for an elderly adult indication will be similar to that of infants. A large-scale efficacy study in adults will likely be required by regulatory agencies even if the vaccine is shown to be effective in infants and children. That requirement will present a huge financial and logistical obstacle since the attack rates for pneumococcal disease are lower in the elderly than in infants and young children. Furthermore, since 23-valent purified pneumococcal polysaccharide vaccine (PPSV23) will be the likely control vaccine, the primary end point is unlikely to be pneumococcal disease caused by serotypes not contained in PPSV23 as very few such cases would be expected. Instead, the study will likely be a comparison of the attack rates for pneumococcal disease caused by any serotype in subjects receiving PPSV23 versus subjects receiving the common protein vaccine.

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47 Polysaccharide-Based Conjugate Vaccines for Enteric Bacterial Infections: Typhoid Fever, Nontyphoidal Salmonellosis, and Escherichia coli O157:H7 Shousun C. Szu, John B. Robbins, Rachel Schneerson, and F.-Y. Lin Program of Developmental and Molecular Immunity, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, U.S.A.

INTRODUCTION Bacterial surface polysaccharides, either as capsules or as Ospecific polysaccharides (O-SPs) expressed by gram-negative organisms, are common virulence factors and essential protective antigens. It is hypothesized that one of the ways that the licensed purified polysaccharide vaccines (e.g., pneumococcal and Salmonella typhi Vi) and conjugate vaccines [e.g., Haemophilus influenzae type b (Hib), pneumococcal, and meningococcal] function is by eliciting a critical level of serum IgG that ‘‘exudes’’ onto the mucosal sites and lyses pathogens upon contact (1). Polysaccharide vaccines are safe and efficacious, and can be administered in multivalent form without interference. For example, the pneumococcal polysaccharide vaccine contains 23 types and meningococcal vaccine contains 4 (2,3). Also proven in field trials, conjugate vaccine-induced antipolysaccharide antibodies inhibit colonization by the target bacteria, thereby resulting in ‘‘herd’’ immunity (4). Purified polysaccharide vaccines are T-independent antigens that do not induce booster response on reinjection, the duration of immunity is short (3–5 years), and they are notoriously poor immunogens for young children. Hib conjugate vaccines showed that the immunogenicity of polysaccharide antigens can be significantly improved by linking the capsular polysaccharide to a carrier protein (5,6). This same principle was applied successfully in the development and licensure of pneumococcal and meningococcal conjugate vaccines (7–9). This approach has also been successfully applied to antigens from enteric pathogens such as Vi polysaccharide, the capsular polysaccharide of S. typhi; a Vi conjugate vaccine was highly immunogenic and protective in young children in an area of high typhoid endemicity (10). For gram-negative pathogens that lack a capsule, the OSP of the outer membrane serves as a virulence and protective antigen. Experimental and clinical data show that immunity in humans following disease is specific to O-SP of Shigella, Escherichia coli, and Vibrio cholerae (11,12). For nontyphoidal Salmonella, mice can be protected from Salmonella typhimurium infection by passive immunization with a monoclonal IgG directed against O-SP (13). Unlike capsular polysaccharides, the O-SPs are normally poorly immunogenic, probably because of their small size; O-SPs need to be conjugated with carrier proteins to enhance their immunogenicity. Our laboratory has applied this

technique to several enteric pathogens including nontyphoidal Salmonella, Shigella, V. cholerae, and E. coli O157. In this chapter, S. typhi, nontyphoidal Salmonella, and E. coli O157 vaccine developments will be reviewed.

TYPHOID FEVER Typhoid fever, which is transmitted to susceptible hosts by food and water contaminated with S. typhi, remains a major health problem in developing countries. Since the 1990s, the emergence of antibiotic resistant strains has made the treatment of typhoid fever more difficult (14). Asymptomatic carriers constitute the reservoir of infection (15). With no near-term solution to providing clean water and environment to all populations in developing countries, vaccination is a costeffective way to control and eliminate this disease (16–18). Typhoid fever in young children is often unrecognized because of atypical clinical symptoms and difficulties in the volume of blood that can be drawn for culture. Recently, using active surveillance methods a high incidence of typhoid fever was shown in young children in densely populated urban slums in South Asia (19). In some surveys in developing countries, children two to four years old had the highest attack rate in both community- and hospital-based surveys (18,19). Currently, there is no licensed typhoid vaccine suitable for this age group. There are three licensed typhoid vaccines, each having limitations. The killed whole-cell parenteral vaccine provides *65% protection but strong adverse reactions limit its usefulness. In the late 1980s and early 1990s, two new and safer vaccines were licensed: the live, attenuated oral vaccine Ty21a and the parenteral administered Vi polysaccharide vaccine. There are major differences between the two vaccines. Vi has consistently provided *65% to 70% efficacy in field trials in highly endemic areas, with protection lasting at least three years (20–22). Ty21a confers a similar level of protection for up to seven years (23–25). Vi immunization consists of a single injection, whereas Ty21a requires three doses. Vi polysaccharide is poorly immunogenic in infants and data for protection do not exist for Ty21a for children less than three years (26). The immunogenicity of Vi vaccine has been substantially improved by conjugation with carrier proteins (10).

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Vi Polysaccharide Vaccine Vi is the capsular polysaccharide of S. typhi, a group D Salmonella. The name Vi was given because the capsule is an essential virulence factor for the pathogenicity of S. typhi. Vi is composed of a (1?4) linked homopolymer of galacturonic acid with N- and O-acetylation at its O2 and O3 positions. The molecular weight of Vi is in the range of 500 kDa to 2000 kDa and is acid and heat stable. These characteristics enable Vi to be a useful vaccine. The protective mechanism of purified Vi is believed to be mediated by serum IgG that inactivates the inoculum upon contact on the epithelial surface. Serum antibodies likely also act upon S. typhi that gain access to the bloodstream. Four efficacy trials in endemic regions: Nepal, South Africa, and China (2) demonstrated that Vi vaccine is safe and conferred *65% to 70% efficacy against S. typhi. Vi vaccine is now licensed in more than 95 countries (20–22). Vi is not protected by patent rights, facilitating technology transfer to developing country producers. The technology for large-scale production of Vi is published in World Health Organization Technical Report Standardization and has been transferred to several countries where typhoid fever is endemic, including India, Vietnam, China, and Indonesia (27). Vi polysaccharide produced in these countries showed comparable safety and immunogenicity as Vi vaccine manufactured by pharmaceutical companies in industrialized countries (22,28). These locally produced Vi vaccines are available at lower prices than imported Vi vaccine in these endemic countries. Furthermore, due to its heat and acid stable nature, dried Vi polysaccharide bulk can be stored at
208C for as long as 10 years and that makes stockpile of the vaccine feasible. Because of its simple chemical composition, the potency of Vi vaccine is regulated by molecular identification, which makes monitoring by regulatory authorities easy and unambiguous (27). For all these reasons, Vi vaccine is considered to be a suitable typhoid vaccine for public health program in developing countries. Nevertheless, Vi vaccine also has limitations: reinjection is recommended every three years; it does not induce booster response upon reinjection; and finally, children under five years respond with low levels of Vi antibodies of shorter duration (29,30). Similar to other polysaccharide conjugate vaccines, these deficiencies were overcome by conjugating Vi to carrier proteins (10).

Vi-rEPA Conjugate Vaccine Vi was conjugated to recombinant exoprotein A (rEPA) from Pseudomonas aeruginosa. A series of clinical studies of this conjugate vaccine confirmed its safety and improved immunogenic properties. The conjugate was shown to be safe and

immunogenic in U.S. adults before clinical trials were undertaken in highly endemic areas in the Mekong Delta, Vietnam, where the annual attack rate for children under five is 0.5% (19,32). Clinical trials in adults, school-age children, and preschool (age 2–4 years) children demonstrated its safety and immunogenicity in the Vietnamese population (33). In schoolage children, the Vi conjugate vaccine elicited significantly higher antibody levels than unconjugated Vi vaccine. A phase III double-blind, placebo-controlled, randomized trial in Vietnam in 11,091 two to five-year old children showed that the conjugate vaccine provided 89% protection over nearly four years of follow-up. The estimated protective level of serum anti-Vi IgG is 3.52 EU (approximately equivalent to 0.11 mg/ mL anti-Vi IgG) (10). The immunogenicity of Vi-rEPA is dosage-dependent; higher dosages elicited higher levels of Vi antibodies (34). Long-term follow-up of immunogenicity showed that adults injected 10 years earlier retained significantly higher levels of anti-Vi IgG than their prevaccination baseline. Vi-rEPA Conjugate Preparation Vi polysaccharide from S. typhi (provided by Sanofi Pasteur, Lyon, France) was covalently linked with purified carrier protein (rEPA) at the National Institutes of Health and designated as Vi-rEPA. Each injection (0.5 mL) contained 25 mg of Vi polysaccharide. Five clinical lots were prepared over the course of our clinical studies, and all elicited similar levels of immune response, indicating consistency in manufacture of Vi conjugates (10,33,34). Phase I and II Clinical Trials in a High Endemic Area Phase I and phase II studies showed that Vi-rEPA was safe and immunogenic in all groups of subjects 2 years of age (Table 1) (33). One injection of the Vi-rEPA in adults elicited a mean of 48-fold rise in serum anti-Vi IgG level six weeks later and remained at a GMT 10-fold above baseline at 26 weeks after immunization (119 EU vs. 9.62 EU; p < 0.0001) (33). The persistence of serum Vi antibody was evaluated again at 3 and 10 years after the injection; the antibody level declined slightly from the value recorded at 26 weeks (92.6 and 68.0 EU vs. 119 EU, p > 0.1). A comparison of the immunogenicity of Vi and Vi-rEPA was conducted in school-age children, 50 in each group. Twenty-six weeks after one injection, the serum anti-Vi IgG level in children who received Vi was 13.4 EU versus 30.6 EU in children who got Vi-rEPA (p < 0.001). To evaluate Vi-rEPA in younger children, 203 children two to four years of age were injected once or twice (6 weeks apart). There were no significant adverse reactions attributable to the vaccine. Six weeks after the first injection, 202/203

Table 1 Serum Anti-Vi IgG in Vietnamese Adults, School-Age Children, and Two- to Four-Year-Old Children Injected with Vi-rEPA Conjugate Anti-Vi IgG (ELISA units) Age (yr)

N

No. of inj.

18–35 5–14 2–4

22 55 48 52 50

1 1 1 2 1

a

    

conj conj conj conj Vi

Blood samples taken and booster shot given. b Anti-Vi IgG at third year versus Pre, p < 0.001.

Pre

6 wka

10 wk

0.5 yr

3 yrb

10 yr

9.62 0.67 0.19 0.18 0.44

465 169 77.2 69.9 18.9

NA NA 54.3 95.4 NA

119 30.0 20.4 30.6 13.4

92.6 14.80 4.83 4.56 NA

68.0 NA

NA

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Table 2 Adverse Reactions in Children Two to Five Years Old Who Received Two Injections of Vi-rEPA Conjugate First injection

N Temp. >37.58C > 39.08C Swelling >5 cm Erythema >5 cm

Vi-rEPA

Placebo

Vi-rEPA

Placebo

5991 81 (1.35%) 17 (0.28%) 0 0

6017 32 (0.53%) 5 (0.08 %) 0 0

5525 109 (1.97%) 1 (0.02%) 20 (0.36%) 2 (0.04%)

5566 25 (0.45%) 1 (0.02%) 1 (0.02%) 0

Table 3 Efficacy of Vi-rEPA in Children Two- to Five-Years Old Over 46 Months of Follow-Up, Including 27 Months of Active Surveillance Followed by 19 Months of Passive Surveillance Variable No. No. No. No.

fully immunized of typhoid cases of single dosea of typhoid cases

Second injection

Vaccine group

Placebo group

Vaccine efficacy (95% CI)

5466 8 388 1

5506 73 383 8

89.0% (76.0–96.9) 87.7% (50.1–94.8)

a

Children received only one injection but participated in the surveillances.

children had a 8-fold rise in serum anti-Vi IgG. There was a booster response after the second dose. Phase III Clinical Trial The safety, immunogenicity, and efficacy of the Vi-rEPA conjugate vaccine were evaluated in a double-blind, randomized trial in children two to five years old in 16 communes in Dong Thap province, Vietnam (10). In 1998, 11,091 two to five-year old children received two injections, six weeks apart, of either Vi-rEPA or a saline placebo. Less than 2% of children had adverse reactions, none considered serious (Table 2). Cases of typhoid, confirmed by the isolation of S. typhi from blood cultures after three or more days of fever, were identified by active surveillance over a period of 27 months, and passive surveillance during an additional 19 months after the vaccine code was opened (9). Over 27 months of follow-up, S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA versus from 47 of the 5566 children who received both injections of placebo (91.5% efficacy, 95% CI, 77.1–96.6) (Table 3). During the 19 months of passive surveillance, typhoid was detected in 3 vaccinees and in 17 placebo recipients (82.4% efficacy; 95% CI, 22.3–99.1). Over the entire 46-month period, the vaccine efficacy was 89.0% (95% CI, 76.0–96.9) (Table 3) (10). Among children who received only one injection (n ¼ 771), there was one case of typhoid in 388 children in the vaccine group and 8 cases in 383 children in the placebo group; thus, the estimated efficacy with only 1 injection of Vi-rEPA was 87.7% (95% CI, 50.1–94.8). Throughout the surveillance period, blood samples were collected monthly from four randomly selected participants of each commune for assessing IgG anti-Vi (10). The persistence of serum IgG anti-Vi level was examined as the geometric means (GMs) for each half-year. There was an age dependence in IgG anti-Vi response, but the difference of GM levels between the younger (2–3 years old) and older (4–5 years old) was not statistically significant. We estimated the protective level of IgG anti-Vi to be 3.52 EU (*0.11 mg/mL IgG) based on the GM of the younger age group at 46 months, since there was no statistically significant difference between the efficacies in the

two stratified age groups (10). At 42 months after vaccination, the GM level of IgG anti-Vi decreased from 22.50 to 3.66 EU in the vaccine group, and increased from 0.65 to 0.80 EU in the placebo group (3.66 vs. 0.8, p < 0.001). The slight increase in the placebo group could reflect the environmental stimulation during the study period. Long-Term Follow-up of Phase III Study Since our ultimate goal for Vi-rEPA trials is to incorporate this vaccine into immunization programs, data on long-term protection and antibody persistence are essential. A follow-up study is underway, which will provide information on the duration of protection in adults and school-age children, and should determine whether a booster dose of Vi-rEPA is necessary. The preliminary data show that in adults, the anti-Vi IgG level remained more than sevenfold higher than the prevaccination baseline 10 years after vaccination. The phase III study populations provide a unique opportunity to assess protection eight years after one or two injections of Vi-rEPA were administered at two to five years of age, and four years after one injection at five to eight years. All children in the phase III trial are now 10 to 13 years old. They represent *7% of the total population of the trial district of this age group (5–15 years old), the peak age incidence of typhoid in Vietnam. The reduction in typhoid fever in this age cohort might have reduced transmission of S. typhi to families and contacts of other age groups. Our review of hospitalized cases of typhoid in all ages from the trial district and nontrial district will provide data on the effects of vaccination on the community. Protection will be assessed by comparing the rates of hospitalized typhoid fever among children in the trials to that in unvaccinated children of similar age in the adjacent district/ town, that is, Binh Thanh district and Cao Lanh town. The persistence of antibody will be assessed by a serological survey in the twice-injected and once-injected children compared with the levels in unvaccinated children of similar age. Serological comparison will also be evaluated for the persistence of IgG anti-Vi in the vaccinated children and compared with the levels in unvaccinated children of similar age. Dosage Study Dosage-related immunogenicity has been observed for other polysaccharide conjugates such as Hib and pneumococcal vaccines. A dosage-immunogenicity study of Vi-rEPA was evaluated in 241 children two to five years old in Phu Tho province, Vietnam. Children were divided randomly into three groups, each received two injections, six weeks apart, containing 25 mg (full dosage), 12.5 mg, or 5 mg of Vi-rEPA. At 10 weeks after the first injection, all children responded with greater than estimated protective level of anti-Vi IgG (>3.52 EU/mL) (Table 4) (34). There was a direct correlation between the dosage and the

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Table 4 Serum Anti-Vi IgG Response in Children Two to Five Years Old Receiving Two Injections of Vi-rEPA at Various Dosages Dosage (mg) of Vi as Vi-rEPA

Number of children, IgG anti-Vi (N, GM of ELISA units/mL)

5.0 12.5 25.0

80, 0.17 80, 0.14 78, 0.13

80, 43.0 80, 74.7 77, 102

75, 6.43 79, 11.3 77, 13.3

11.3 and 13.3 versus 6.43, p < 0.001; 13.3 versus 11.3, p > 0.5.

immune response. At one year’s time, the anti-Vi IgG levels were 13.3, 11.3, and 6.43, respectively (13.3 and 11.3 vs. 6.43, p < 0.001; 13.3 vs. 11.3, NS). The anti-Vi IgG levels in all three dosages declined about sevenfold from the 10 weeks levels, but remained significantly higher than the preimmune levels (p < 0.0001); 96% of children still had levels eightfold or greater over their baseline. Based on these data, we recommend a dosage of 25 mg for all ages. This study also confirmed the safety and consistent immunogenicity of the four lots of Vi-rEPA used in this and previous trials (10,33,34). Infant Study To complete the full course of clinical studies of the Vi conjugate vaccine, a safety and immunogenicity study of Vi-rEPA administered to infants concurrently with routine immunizations (DPT, polio, and hepatitis B) is currently under study in Phu Tho province, Vietnam. In this study, 308 infants were randomly divided into three groups, each receiving four injections of VirEPA or Hib conjugate vaccine or none, in addition to the routine vaccines at 2, 4, 6, and 12 months. Safety data compared among groups showed that Vi-rEPA was safe in infants. Cord blood at birth will be used for estimation of preimmune anti-Vi levels. Blood samples taken at 7, 12, and 13 months will be analyzed and compared for their DPT, Vi, or Hib IgG levels, whenever applicable. The level of anti-Vi IgG will be compared with the estimated protective level from our phase III efficacy trial for assessment of the protection against typhoid fever.

Vi as a Probe to Investigate the Effect of Birth Weight on Antibody Responses in Adulthood There is evidence that links low birth weight to susceptibility to chronic disease in adulthood. Evidence is also emerging that some components of immune function may be programmed in early life. The relation between size at birth (full term) and response to Vi vaccination in a cohort of 257 adults (mean age: 29.4 year; 146 men) born in an urban slum in Lahore, Pakistan, during 1964 to 1978 was studied. A single dose of Vi polysaccharide vaccine or two doses of rabies vaccine, representing Tindependent and T-dependent antigens, respectively, were given to the volunteers. Antibody titers were measured in pre- and postvaccination serum samples. Response to typhoid vaccination was positively related to birth weight (IgG anti-Vi; r ¼ 0.138, p ¼ 0.031; IgM anti-Vi, r ¼ 0.197, p ¼ 0.034) but no correlation was found with the rabies vaccine (35). Reinjection of Vi three years later was compared with another T-dependent antigen, the Hib conjugate. The results showed the same birth weight dependence for Vi but not for Hib conjugate (36). These findings add to a growing body of evidence suggesting that components of the immune system may be permanently programmed by events in early life. The contrasting effects on responses to typhoid and rabies or Hib conjugate vaccines suggest that antibody generation to

polysaccharide antigens is compromised by fetal growth retardation. Since this birth weight phenomenon was not observed with a polysaccharide conjugate vaccine, the early-life programming is probably targeted mainly at B-cell immunity. It still remains the case that the molecular basis of immune responses to polysaccharide antigens is the least well understood of all antibody responses yet clinically deficient responses have the most profound consequences for humans. We plan to use the Vi vaccine as a probe to assess functional immunity in a cohort of children five years old in Gambia, which might help us to understand conditions in infancy that affect the immune response in childhood. This particular group is well characterized with detailed information available on maternal nutritional status, fetal growth (by serial ultrasonography), birth size, infant feeding status, growth and morbidity in infancy, and thymic development. The study will link the correlation of birth weight to immune response in early ages.

VACCINES AGAINST SALMONELLA PARATYPHI A AND NONTYPHOIDAL SALMONELLA For many years in South and Southeast Asia, the second most common cause of enteric fever has been Salmonella paratyphi A (37,38). Since 1999 after implementing Vi vaccination, more S. paratyphi A than S. typhi strains have been isolated in the province of Guangxi, southeastern China (39). There has also been an increase in S. paratyphi A infections in the Indian subcontinent (40). In developed countries, the most common causes of Salmonella outbreaks are S. typhimurium (group B) and S. enteritidis (group D) (41). These Salmonella are zoonotic, and the infections are generally foodborne. S. typhimurium and S. enteritidis are also important causes of bacterimia in African children (42). The emergence of multidrug-resistant Typhimurium phage type DT104 in United Kingdom in the 1980s raised alarm over the usage of antibiotics in animal feed. DT104 infection is common in a broad range of food animals, such as poultry, pigs, and sheep (43). DT104 spread to other parts of the world in the 1990s, and it is now a common Salmonella type in as many as 30 countries, mostly industrialized, including the United States, the United Kingdom, Germany, Denmark, and France (43,44). This phage type has become a matter of concern because of its rapid international dissemination and its ability to readily acquire additional resistance traits to other, clinically important antimicrobial drug classes, such as fluoroquinolones, trimethoprim, and cephalosporins (44). There are no licensed vaccines for nontyphoidal Salmonella. The killed whole-cell parenteral TAB vaccine, composed of inactivated S. typhi, S. paratyphi A and B, was discontinued from manufacture in the United States and other countries because of the high rates of adverse reactions and lack of efficacies for the Paratyphi components (45). The major challenges in development of new S. paratyphi A and S. typhimurium vaccines are to reduce adverse reactions and improve immunogenicity. The O-SP of groups A, B, and D Salmonella share the same backbone structure: 2-aD-Manp-(1?4)-aL-Rhap-(1?3)-aD-Galp(1? 3 : 1 R

Chapter 47: Polysaccharide-Based Conjugate Vaccines for Enteric Bacterial Infections

The serogroup specificity resides on the sidechain R, the 3,6,dideoxyhexose branch linked (1?3) to the mannose: paratose for group A, abequose for group B and tyvelose for group D. The rhamnose is partially O-acetylated at C-3 for S. paratyphi A. The essential role of the O-acetyl groups in the immunogenicity of the O-SP in group A has been demonstrated (46). This immunodominant antigenic site is susceptible to chemical treatment and could be inadvertently lost in vaccine preparation. Similarly, in S. typhimurium, the O-acetyl groups are essential in characterizing the organism: both O-acetyl positive and negative strains can cause disease, but they possess distinctive serological traits and the cross reactions between them are limited (13,44).

Conjugate Vaccine for Salmonella paratyphi A The vaccine development for S. paratyphi A in our laboratory is based on the fact that the O-SP antibodies are bactericidal and can lyse inocula upon contact, thereby conferring immunity (46). In general, O-SPs alone are poorly immunogenic; therefore, conjugation with a protein carrier was undertaken. O-SP was purified from the LPS, detoxified by acetic acid to remove the lipid A and covalently linked to tetanus toxoid as the carrier protein (SPA-TT). Phase I and phase II clinical trials of SPA-TT were conducted in an endemic area in southern Vietnam (47). There were no serious adverse reactions observed in any age group tested. The levels of preexisting LPS antibodies were similar in teenagers and adults. Young children had lower preexisting anti-LPS levels but the difference was not statistically significant (Table 5). After one injection, 75% of adults, 85% of teenagers, and 90% of young children responded with fourfold or greater rise in anti-LPS IgG. There was no booster response observed in children receiving two injections; six months after the first injection, the antibody levels remained 3.8 to 4 times higher than the preimmune levels. The antibodies elicited by SPA-TT were bactericidal against S. paratyphi A in vitro. A phase III clinical trial to evaluate the conjugate vaccine in an endemic area in Southeast Asia is being planned.

Salmonella typhimurium Conjugate Vaccine If the S. paratyphi A conjugate proves to be safe and protective, O-SP conjugates of serogroups B, C, and D Salmonella could also be prepared as a strategy to provide protection against nontyphoidal infections. Mouse immunization and challenge models are valid animal models for S. typhimurium. Using active immunization with synthetic conjugate vaccine or by passive immunization with monoclonal antibodies against the O-SP, Svenson and colleagues demonstrated that O-SPs are protective antigens (13,46). Watson et al. (48) also showed that by Table 5 LPS Serum Antibodies Elicited by S. paratyphi A OSpecific Polysaccharide-TT Conjugate Anti-LPS IgG (ELISA units)

Age (yr)/No. of injections

N

0 day

42 daysa 70 days

180 days

18–44/1 13–17/1 2–4/1 2–4/2a

20 108 63 47

1.47 1.69 0.91 0.77

18.5 15.1 19.3 16.7

6.00 7.05 3.47 4.08

a

– – 11.7 11.9

Serum sample taken before the second dose injected.

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conjugating S. typhimurium O-SP to tetanus toxoid, mice immunized injected subcutaneously developed IgG that was bactericidal and protected the mice from subsequent challenge. These investigators demonstrated a 160-fold increase in the 50% lethal dose of S. typhimurium (48) in immunized animals. It is therefore feasible that a modern combination vaccine of TAB conjugates could be formulated.

ESCHERICHIA COLI O157:H7 Enterohemorrahagic E. coli (EHEC) infections are the leading cause of E. coli–induced death and renal failure in industrialized countries. Serious disease and complications can follow EHEC infection, including hemorrhagic colitis, life-threatening hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura. Approximately 5% to 10% of EHEC diarrheal infections in children <5 years of age lead to HUS, a serious complication that is the major cause of acute renal failure and of long-term kidney damage in U.S. children (49,50). Mortality occurs in *5% of HUS patients, and severe outcomes such as permanent renal impairment may follow among survivors (49). E. coli O157:H7 is the most common (albeit not the only) serotype for this pathotype of E. coli and has caused large outbreaks. In 2000, an outbreak of E. coli O157 in Ontario, Canada, from a contaminated water source affected thousands of people (51). In Osaka, Japan, an outbreak led to more than 5000 illnesses and 20 deaths (52). Recently, there were more than a score of outbreaks yearly caused by E. coli O157 in the United States and Canada alone. The treatment of E. coli 0157: H7 infection remains controversial. Antimicrobial agents have not been proven to impact favorably on the severity of disease, duration of shedding, or development of HUS (53); antibiotic therapy of EHEC diarrheal illness may be a risk factor for development of HUS. Because there is no effective treatment for E. coli O157:H7 and also because of the wide range of sources of infection (ingestion of beef, dairy food, vegetables, swimming in contaminated water, visiting petting zoos, etc.), efforts have been directed toward prevention of infection by food inspection and environmental improvements. Since the risk of acquiring E. coli O157:H7 disease is greatest in children <5 years of age, there may be an agerelated acquisition of natural immunity stimulated through contact with homologous or cross-reactive organisms in the environment. In the United States, young children had significantly lower anti-O157 LPS IgG than adults, and also than Vietnamese children and adults (unpublished data). The antibodies detected in adults could be stimulated by cross-reactive organisms in the environment, including Citrobacter freundii (54). Our vaccine preparation is aimed at stimulating high titer serum LPS IgG that is bactericidal. We hypothesize that lysis of the inoculum by means of anti-O157 antibodies should prevent the establishment of the infection and clinical disease. A major virulence factor of EHEC is Shiga toxin (Stx). There are a number of other serotypes of E. coli that express Shiga toxin and cause EHEC (55). For example, in Australia, the dominant EHEC serotype is E. coli O111. There are two major types of Stx, Stx1 and Stx2. Most EHEC disease in the United States is caused by strains secreting Stx2, alone or along with Stx1. Since there is no effective treatment of HUS, passive immunization with antitoxin against Stx1 and Stx2 is being explored as a therapeutic intervention. This also raises the

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possibility that elicitation of Shiga antitoxin can also play a role in prophylactic immunization of high-risk subjects. Similarly, since cattle constitute the main animal reservoir for E. coli O157: H7, anti-EHEC veterinary vaccines are being evaluated, along with other measures, to diminish the extent of the EHEC reservoir among cattle. Vaccine development activities against E. coli O157:H7 can thus be considered as being approached from the following three directions:

1. 2. 3.

Development of an LPS-based E. coli O157:H7 vaccine suitable for children <5 years(56–58) Inclusion of a nontoxic mutant Stx2 molecule or its Bsubunit in the LPS-based vaccine to prevent severe disease caused by Stx2-producing EHEC (59) Development of a vaccine to eliminate E. coli O157 carriage among cattle, thereby eliminating this major reservoir of O157:H7 pathogens

Escherichia Coli O157:H7 Conjugate Vaccine for Children The O-SP of E. coli O157 is a homopolymer composed of a tetrasaccharide repeating unit: 3)-aDGalpNAc-(1?2)-aDPerpNAc (1?3)-aLFucp(1?4)-bDGlcp(1?). To prepare the polysaccharide-protein conjugate, the LPS was detoxified by acid or base treatment and covalently bound to P. aeruginosa rEPA, designated as O157-rEPA. In a phase I study, the safety and immunogenicity of LPSbased E coli O157:H7 conjugates prepared with three different schemes were compared in 87 adult volunteers (56). The vaccines were safe, and elicited high levels of anti-LPS IgG with bactericidal capacity. Most volunteers (81%) responded with more than fourfold increase in LPS IgG one week after vaccination; all volunteers responded with a more than fourfold rise at four weeks, and this level was sustained for at least 26 weeks after injection (Table 6). All three vaccines elicited high titers of serum bactericidal activity that roughly correlated with the serum IgG and IgM LPS antibodies. We continued our investigation of this vaccine in the target age group of young children. Fifty children age two to five years were injected once or twice (6 weeks apart) (57), none of who developed fever or significant local reactions. Nearly all children (98%) responded with 10-fold rise of LPS IgG six weeks after the first injection. The level of LPS IgG remained significantly higher than the preimmune levels six months later (22-fold rise). Reinjection of the conjugate did not induce a booster response as observed with several other conjugate vaccines at this age (57). The conjugate also elicited high titers of serum bactericidal activity that correlated with the serum LPS IgG levels. The antibody response was age related, as adults responded with approximately fourfold higher LPS IgG levels than children. The level of postimmune LPS IgG antibody in the two- to five-year olds was significantly higher than the preimmune levels in adults (6.08 vs. 0.47, p < 0.001).

Table 7 Bactericidal and Neutralization Activities in Sera from Mice Injecteda with E. coli O157:H7 O-SP Conjugated with Stx1B

Immunogen

Anti-LPS IgG (GM, ELISA units)

Bactericidalb

Stx1 neutralizationc

O-SP-Stx1B (AH) O-SP-Stx1B Saline

220 251 <10

50 200 <10

8040 14,400 <100

a Female mice, five weeks old, 10/group, injected IM without adjuvant three times. Anti-LPS expressed in geometric mean ELISA units. b Reciprocal of the highest dilution yielding 50% killing. c HeLa cells monolayer assay; result expressed as the reciprocal of the highest dilution yielding 50% neutralization.

Our objective is to incorporate this vaccine into infant routine immunization programs. Thus, the safety and immunogenicity of this vaccine in infants when administered together with DTP vaccines will have to be studied.

Conjugate Vaccine with Stx as the Carrier Protein An EHEC vaccine may provide broader coverage if it includes Stx or StxB antigens in the formulation, since there are a number of serotypes of E. coli other than O157:H7 that express Stx and cause EHEC disease, including HUS (55). There may also be a synergistic effect in protection if more than one antigen from E. coli O157:H7 is included in the vaccine. Stx1 B subunit can be genetically reconstructed and expressed in V. cholerae in large quantity. StxB can be used as a carrier in conjugation with O-SP of E. coli O157:H7. An example of such conjugate, O157-Stx1B, elicited in mice both anti-LPS IgG and anti-Stx1 IgG with high titers of bactericidal activities and neutralization titer against holotoxin Stx1 in HELA cell assay (GM neutralization titers 16,000 vs. 10 for the control serum) (Table 7) (59). Since the most common type of Stx in HUS disease isolates is Stx2, an optimal vaccine to prevent E. coli O157:H7 infection should include both Stx1 and Stx2 as the carrier component.

Veterinary Vaccine Against E. Coli O157:H7 in Cattle Many outbreaks of E. coli O157 can be traced back to contamination from cattle shedding. Cattle can harbor this organism without symptom for as long as six weeks. During this period, fecal shedding from cattle contaminates soil and water sources in the nearby areas. Eliminating E. coli O157:H7 in cattle as a preslaughter intervention is a means to reduce a major reservoir from which the organism eventually makes its way into human food sources (60). The colonization site of E. coli O157 in cattle has been identified to be in the rectum. Several approaches have been attempted in cattle vaccine: a killed whole-cell vaccine, immunization with intimin or flagella, and a LPS-based parental

Table 6 Serum Anti-LPS IgG in Adults and Children Two to Five Years Old Receiving One or Two Injections of E. coli O157 O-SP-rEPA Conjugate Vaccine IgGa (GM, ELISA units)

IgM (GM, ELISA units)

Age (yr)

N

Pre

6 Mo

Fold rise

Pre

6 Mo

Fold rise

18–44 2 to 5

29 24

0.47 0.27

32.8 5.63

70 21

8.10 2.28

28.6 3.66

3.5 1.6

0.47 versus 0.27, 8.10 versus 2.28, p < 0.01; 32.8 versus 5.63, 28.6 versus 3.66, p < 0.001.

a

Chapter 47: Polysaccharide-Based Conjugate Vaccines for Enteric Bacterial Infections

vaccine (60–62). In collaboration with Dr. Bohach at University of Idaho and Fort Dodge Animal Health Inc., we prepared LPS conjugate vaccine with veterinary tetanus toxoid. Cattle injected with the conjugates had elevated levels of serum anti-LPS IgG and a booster response upon reinjection. Cattle immunized with this conjugate were challenged with 107 colony-forming units of E. coli O157:H7. An inverse correlation was observed between the serum anti-LPS IgG and the level of rectal colonization (unpublished data). Since the duration of immunity was approximately six weeks, this vaccine has the potential to be useful in cattle in feedlots. There is still a need to improve the LPS conjugate vaccine for longer duration of protection for cattle.

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50. Tarr PI. Escherichia coli O157:H7: clinical, diagnostic, and epidemiological aspects of human infection. Clin Infect Dis 1995; 20:1–8. 51. Waterborne outbreak of gastroenteritis associated with a contaminated municipal water supply, Walkerton, Ontario, May to June 2000. Can Commun Dis Rep 2000; 20:170–173. 52. Michino H, Araki K, Minami S, et al. Massive outbreak of Escherichia coli O157:H7 infection in schoolchildren in Sakai City, Japan, associated with consumption of white radish sprouts. Am J Epidemiol 1999; 150:797–803. 53. Wong CS, Jelacic S, Habeeb RL, et al. The risk of the hemolyticuremic syndrome after antibiotic treatment of Escherichia coli O157: H7 infections. N Engl J Med 2000; 342:1990–1991. 54. Nishiuchi Y, Doe M, Hotta H, et al. Structure and serologic properties of O-specific polysaccharide from Citrobacter freundii possessing cross-reactivity with Escherichia coli O157:H7. FEMS Immunol Med Microbiol 2000; 28:163–171. 55. Ludwig K, Bitzan M, Zimmermann S, et al. Immune response to non-O157 Vero toxin-producing Escherichia coli in patients with hemolytic uremic syndrome. J Infect Dis 1996; 174:1028–1039. 56. Konadu EY, Parke JC Jr., Tran HT, et al. Investigational vaccine for Escherichia coli O157: phase 1 study of O157 O-specific polysaccharide-Pseudomonas aeruginosa recombinant exoprotein a conjugates in adults. J Infect Dis 1998; 177:383–387. 57. Ahmed A, Li JP, Shiiloach Y, et al. Safety and immunogenicity of Escherichil coli O157 O-specific polysaccharide conjugate vaccine in 2 to 5-year-old children. J Infect Dis 2006; 193:515–521. 58. Ashkenazi S, Passwell JH, Harlev E, et al. Safety and immunogenicity of Shigella sonnei and Shigella flexneri 2a O-specific polysaccharide conjugates in children. J Infect Dis 1999; 179:1565–1568. 59. Konadu E, Donohue-Rolfe A, Calderwood SB, et al. Syntheses and immunologic properties of Escherichia coli O157 O-specific polysaccharide and Shiga toxin 1 B subunit conjugates in mice. Infect Immun 1999; 67:6191–6193. 60. Van Donkersgoed J, Hancock D, Rogan D, et al. Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan. Can Vet J 2005; 46:724–728. 61. Johnson RP, Cray WC, Johnson ST. Serum antibody responses of cattle following experimental infection with Escherichia coli O157: H7. Infect Immun 1996; 64:1879–1883. 62. Potter AA, Klashinsky S, Li Y, et al. Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins. Vaccine 2004; 22:362–369.

48 Attenuated Strains of Salmonella enterica Serovars Typhi and Paratyphi as Live Oral Vaccines Against Enteric Fever Myron M. Levine, James E. Galen, Marcela F. Pasetti, and Marcelo B. Sztein Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

TARGET POPULATIONS Typhoid fever caused by Salmonella enterica serovar Typhi (S. Typhi) and paratyphoid fever caused by Salmonella enterica serovar Paratyphi (S. Paratyphi) A or B are exceedingly uncommon in modern industrialized countries where populations are served by treated, bacteriologically monitored water supplies and by sanitation that removes human fecal waste. In contrast, in many less developed countries where segments of the population commonly lack such amenities, typhoid and paratyphoid fevers (referred to as enteric fevers) are often endemic. From the public health perspective, enteric fevers typically constitute the most important enteric disease problem of school-age children (1). Systematic clinical, epidemiologic, and bacteriologic surveillance for typhoid fever carried out in relation to field trials of efficacy of candidate vaccines and as assessments of disease burden has provided more precise data on the incidence of typhoid and paratyphoid fever in many populations. Many of these studies relied on passive surveillance that detected cases severe enough for the patient (or the patient’s caretaker) to seek health care (2–8); other studies used forms of active surveillance (9–12). The incidence rates recorded were much higher than predicted on the basis of nonsystematic surveillance. In urban slum environments in South Asia, systematic household and health center–based active surveillance demonstrated a high incidence of bacteremic typhoid infection among febrile toddlers and preschool children (13,14). Besides children and young adults in less developed countries, travelers (15–17) and clinical microbiologists (18,19) represent other groups at increased risk of developing typhoid and paratyphoid fever. Among U.S. travelers, the risk is highest in the Indian subcontinent (16,17). Because of increased exposure to S. Typhi and S. Paratyphi A and B, clinical microbiologists, even in industrialized countries, constitute an identifiable group at increased risk of developing enteric fever (18,19).

MULTIRESISTANT SALMONELLA TYPHI AND SALMONELLA PARATYPHI STRAINS Since the 1990s, strains of S. Typhi and S. Paratyphi A resistant to most of the antimicrobials that were previously clinically effective have spread aggressively throughout the Middle East, the Indian subcontinent, and Southeast Asia (20–24). The few antibiotics that remain effective against these multiply resistant

strains, such as ciprofloxacin, ceftriaxone, and azithromycin, are relatively expensive and not readily available in rural areas of less developed countries. The dissemination of these multiply resistant S. Typhi and S. Paratyphi A strains in many less developed countries has rekindled interest in the development of improved oral typhoid vaccines.

LICENSED LIVE ORAL TYPHOID VACCINE Ty21a: ATTRIBUTES AND LIMITATIONS Ty21a, an attenuated strain of S. Typhi that is safe and protective as a live oral vaccine, was developed in the early 1970s by chemical mutagenesis of pathogenic S. Typhi strain Ty2 (25). This pioneering vaccine strain has many attributes but also has several notable deficiencies. The characteristic mutations in Ty21a include an inactivation of galE (which encodes uridine diphosphate-galactose-4-epimerase, an enzyme involved in lipopolysaccharide [LPS] synthesis) and an inability to express Vi capsular polysaccharide. However, Ty21a also harbors more than two dozen additional mutations compared with its wildtype parent. Whereas Ty21a was remarkably well tolerated in placebo-controlled prelicensure clinical trials and in extensive postlicensure surveillance (26,27), it is not clear exactly which mutations are collectively responsible for the stable attenuated phenotype of this vaccine. On the other hand, it is known that mutations in galE and loss of Vi expression alone cannot account for the attenuation because a recombinant vaccine candidate (EX 462) made from the same wild-type parent (Ty2) with precise mutations in galE and via was not attenuated when fed to healthy adult volunteers (28). Ty21a provides significant protection without causing adverse reactions. Results of three double-blind, placebocontrolled studies that utilized active surveillance to assess the reactogenicity of Ty21a in adults and children show that adverse reactions were not observed significantly more often in vaccinees than placebo recipients for any symptom or sign (5,29,30). In large-scale efficacy field trials with Ty21a, involving approximately 514,150 schoolchildren in Chile (2–4), 32,388 in Egypt (31), and approximately 20,543 subjects from three years of age to adults in Indonesia (5), passive surveillance failed to identify vaccine-attributable adverse reactions (2–5,32). Results of controlled field trials of Ty21a revealed that the formulation of the vaccine, the number of doses administered,

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and the spacing between the doses markedly influence the level of protection that can be achieved (1–5,31,33,34). In the first field trial of Ty21a in Alexandria, Egypt, six- to seven-year-old schoolchildren received three doses of vaccine (suspended in a diluent) on Monday, Wednesday, and Friday of one week (31); to neutralize gastric acid, the children chewed a 1.0-g tablet of NaHCO3 several minutes before ingesting the vaccine or placebo. During three years of follow-up, 96% protective efficacy against bacteriologically confirmed typhoid fever was observed (31). A practical formulation that has been a commercial product since the mid-1980s consists of lyophilized vaccine in enteric-coated, acid-resistant, capsules (1,2). In a randomized, placebo-controlled field trial in Santiago, Chile, three doses of this enteric-coated formulation given within one week provided 67% efficacy during the first three years of follow-up (2) and 62% protection over seven years of follow-up (35). Four doses of Ty21a in enteric-coated capsules given within eight days are significantly more protective than two or three doses (33). When the enteric-coated capsule formulation of Ty21a was licensed in the United States by the Food and Drug Administration in late 1989, it was with a recommended schedule of four doses given at an every other day interval; other countries use a three-dose immunization schedule. In the mid-1980s, the Swiss Serum and Vaccine Institute (currently Berna Biotech, a Crucell Company) succeeded in preparing for large-scale field trials of a ‘‘liquid suspension’’ formulation of Ty21a that was amenable to large-scale manufacture. The new formulation consisted of two packets, one containing a dose of lyophilized vaccine organisms and the other containing buffer. To prepare a ‘‘vaccine cocktail,’’ contents of the two packets were mixed in a cup containing 100 ml of water and the resultant suspension was then ingested by the subject to be vaccinated. Another randomized, controlled field trial undertaken in Santiago, Chile (4) and a parallel trial carried out in Plaju, Indonesia (5) directly compared this new liquid formulation (that somewhat resembles what was used in the Alexandria, Egypt field trial) with the enteric-coated capsule formulation. In both the trials in South America and Asia, Ty21a administered as a liquid suspension was superior to vaccine in enteric-coated capsules; in the Santiago trial the difference was statistically significant (4). Moreover, Ty21a given as a liquid suspension protected young children as well as older children (4,5). In previous trials with enteric-coated vaccine, young children were not as well protected as older children (2). The liquid formulation was more practical for giving Ty21a to children <7 years of age and was strongly immunogenic in toddlers and preschool children (36,37). In contrast, an attempt to prepare a ‘‘simple’’ liquid suspension of Ty21a by emptying the contents of an enteric-coated capsule into milk containing 0.5 g of NaHCO3 resulted in an ineffective mixture that was poorly immunogenic (38). Despite its pioneering role and many positive attributes (including excellent safety record, clinical acceptability, minimal reactogenicity, and stimulation of a moderate level of long-lived protection), recognized drawbacks of Ty21a include the lack of a molecular basis for its attenuation, relatively modest immunogenicity, and, most importantly, the need to administer at least three spaced doses to confer a moderate level of protection of extended duration (2,3,35). Accordingly, a new generation of live oral typhoid vaccines aims to be as well tolerated clinically as Ty21a, yet more immunogenic and protective, and to require administration of just a single oral dose.

Correlates of Protection of Ty21a Although Ty21a is only modestly immunogenic and requires three or four spaced doses to elicit protection, the efficacy is surprisingly long lasting, enduring for at least five to seven years (35). Two immunologic assays were found to correlate with the protection conferred by different formulations and immunization schedules of Ty21a in field trials. These include serum IgG O antibody seroconversions (34) and enumeration of gut-derived IgA O antibody-secreting cells (ASCs) detected among peripheral blood mononuclear cells (39). The identification of these measurements as immunologic correlates of protection provided invaluable tools for use in clinical trials of new attenuated S. Typhi strains as possible live oral vaccines. More recently, it has been found that oral Ty21a is a potent stimulator of CD8þ cytotoxic T cells (CTLs) that recognize cellular targets infected with S. Typhi, as well as interferong-producing T cells (40,41). It is surmised that these T-cell responses also contribute to and correlate with protection, but this cannot be proven, since these assays were not available at the time of the field trials of Ty21a.

A NEW GENERATION OF ATTENUATED SALMONELLA TYPHI LIVE ORAL VACCINES Investigators in various laboratories worldwide have undertaken to engineer new candidate vaccine strains that are as well tolerated as Ty21a yet more immunogenic, such that a single oral dose will elicit long-lived protective immunity. One early attempt was made to increase the immunogenicity of Ty21a by restoring its ability to express Vi antigen by introducing viaB (which encodes the enzymes required for synthesis of Vi polysaccharide) from wild-type strain Ty2 into the chromosome of Ty21a and demonstrating expression of Vi (42,43). Whereas the Vi-positive variant was well tolerated when fed to adult volunteers and most subjects who received three doses developed rises in serum IgG antibodies and IgA ASCs against S. Typhi O antigen, no subject manifested a rise in serum IgG anti-Vi or exhibited ASCs that secrete IgA anti-Vi (43).

Importance of the Wild-Type Parent Strain Of the various attenuated vaccine strains that have been evaluated in clinical trials during the past two decades, each was derived from one of three wild-type parent strains, including Ty2, ISP1820, and CDC 1080. The choice of wild-type parent selected for attenuation influences the characteristics and nature of the ultimate live vaccine strain produced. Evidence for this derives from the different clinical acceptability of the resultant vaccine strains when identical mutations were introduced into distinct wild-type backgrounds (e.g., aroC and aroD into Ty2 vs. ISP1820).

Attenuating Strategies Candidate vaccine strains have been prepared by inactivating genes encoding various biochemical pathways (28,44–47), global regulatory systems (48), heat shock proteins (49), other regulatory genes (50–52), and putative virulence properties (53–55). Elucidation of the sequences of the complete genomes of wildtype S. Typhi strain Ty2 (56) and CT 18 (57) has facilitated the development of attenuated S. Typhi strains. The relative attenuating potential of various mutations has often been assessed preliminarily by feeding S. Typhimurium strains harboring these mutations to mice and observing the

Chapter 48: Attenuated Strains of S. Typhi and S. Paratyphi as Live Oral Vaccines

result in comparison with isogenic wild-type strains. However, as clinical information with various engineered strains has accrued, it has become evident that the behavior of attenuated S. Typhimurium strains in mice does not adequately predict the behavior of homologous S. Typhi mutants in humans (58). Several examples can be cited where specific mutations that successfully attenuated S. Typhimurium for mice failed to adequately attenuate S. Typhi for humans (28,59,60). These observations underscore the critical importance of evaluating candidate live oral S. Typhi vaccine strains in carefully designed and executed clinical trials.

541Ty and 543Ty Stocker et al. pioneered the concept of making auxotrophic mutants of Salmonella by inactivating genes encoding enzymes in the aromatic amino acid biosynthesis pathway (44,45), a strategy subsequently adopted and modified in the design of multiple other attenuated S. Typhi vaccine candidates that have been evaluated in clinical trials (54,59,61–64) (Table 1). For this reason, even though Stocker’s original constructs (541TY and 543Ty) did not progress beyond phase I and were only minimally immunogenic, they are described here in some depth. Mutations in various aro genes described by Stocker render invasive Salmonella serovars nutritionally dependent on substrates (2,3 dihydroxybenzoate and para-aminobenzoic acid) that are not available in sufficient quantity within mammalian tissues; as a consequence, the vaccine strains remain viable but are severely inhibited in their ability to proliferate in the intracellular environment. Edwards and Stocker (45) constructed prototype strains 541Ty and 543Ty (a Vi-negative variant of 541Ty) from CDC 1080, a wild-type strain obtained from the collection of the Centers for Disease Control and Prevention (CDC). It may be of some relevance that the pathogenicity of this strain had never been directly tested in volunteers. In contrast, several other

499

groups of investigators started with wild-type strain Ty2, the parent of Ty21a (25), in their attempts to engineer new attenuated strains (51,54,61,65). The pathogenicity of Ty2 was unequivocally established in experimental challenge studies in volunteers performed several decades ago (66), and this strain was thereupon used as the challenge organism to assess the efficacy of various live oral (67–69) and nonliving oral (70) typhoid vaccines. Strains 541Ty and 543Ty also harbored a deletion mutation in purA, which results in a specific requirement for adenine (or an assimilable compound such as adenosine). A third mutation in hisG, leading to a histidine requirement, does not affect virulence but provided an additional biochemical marker to clearly differentiate the vaccine strain from wild S. Typhi. Strains 541Ty and 543Ty were quite well tolerated in dosages up to 5  1010 colony-forming units (CFU) in phase I studies but were notably less immunogenic than Ty21a in stimulating humoral antibody responses (46). For example, only 11% of subjects developed serum IgG anti-O antibodies.

Attenuated Salmonella Typhi Strain CVD 908 The first vaccine strain that proved to be well tolerated and impressively immunogenic following administration of a single oral dose in phase I clinical trials in humans is strain CVD 908 (60,62,71), which harbors precise deletion mutations in aroC and aroD (61) in the Ty2 background (Table 1). At a well-tolerated dose of 5  107 CFU, 92% of CVD 908 recipients manifested IgG O antibody seroconversions and showed evidence of priming of the intestinal immune system (IgA ASCs) (62). Moreover, vaccinees exhibited lymphoproliferative responses, and their PBMCs were shown to secrete cytokines (in particular, interferon-g) upon exposure to S. Typhi flagella (72). CVD 908 also stimulates cytotoxic lymphocytes that recognize targets (Epstein-Barr virus–immortalized prevaccination B lymphocytes) expressing S. Typhi antigen on their surface (73).

Table 1 Attenuating Mutations Present in Recombinant Strains of Salmonella Typhi That Have Been Evaluated in Clinical Trials as Candidate Live Oral Vaccines Vaccine strain

Wild-type parent

Clinical phenotype

Immunogenicity phenotype

References

EX645 541Ty 543Ty

Ty2 CDC 1080 CDC 1080

Not attenuated Overly attenuated Overly attenuated

Immunogenic Poorly immunogenic Poorly immunogenic

28 45,46 45,46

CVD 906 CVD 908

ISP 1820 Ty2

Immunogenic Highly immunogenic

59 60–62

CVD 908htrA CVD 909

Ty2

Insufficiently attenuated Attenuated (but silent bacteremias at high dosage levels) Attenuated

Immunogenic

49,79,81

Ty2

Attenuated

Immunogenic

84

PBCC211 PBCC222

CDC 1080 CDC 1080

X3927 X4073 X8110

Ty2 Ty2 ISP1820

Immunogenic Poorly immunogenic at welltolerated dosage levels Immunogenic Immunogenic Weakly immunogenic

64 64

cya, crp cya, crp, cdt cya, crp, cdt

48,60,65 65,87,88 89

phoP/phoQ phoP/phoQ, aroA aroC, ssaV

Ty800 Ty445 M01ZH09

Ty2 CDC 1080 Ty2

Insufficiently attenuated Insufficiently attenuated at high dosage level Insufficiently attenuated Attenuated Attenuated (but silent bacteremias at high dosage levels) Attenuated Overly attenuated Attenuated

Immunogenic Poorly immunogenic Immunogenic

51 63 54

Mutated gene galE, via aroA, purA aroA, purA, Vi-negative aroC, aroD aroC, aroD aroC, aroD, htrA aroC, aroD, htrA; Ptac-tviA aroA, aroD aroA, aroD, htrA

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One drawback observed in the phase I clinical trials with CVD 908 is that 50% of subjects who ingested this vaccine strain at a dose of 5  107 CFU (62) and 100% of subjects who received a 5  108 CFU dose (74) manifested clinically silent vaccinemias, wherein vaccine organisms were recovered from blood cultures collected at one or more time points between day 4 and 8 after vaccination. The blood cultures were collected systematically in these individuals within hours after they ingested vaccine and then on days 2, 4, 5, 7, 8, 10, 14, 20, 27, and 60. No blood culture from any vaccinee was positive prior to day 4, nor after day 8. The vaccinemias appeared to have no clinical consequence (for example, they were not associated with fever) and were short lived, spontaneously disappearing without the use of antibiotics. These clinical results posed a vaccinology dilemma. There is precedent for the licensure and widespread use in public health of live vaccine strains that cause shortlived, self-limited vaccinemia. For example, viremia occurs in many recipients of attenuated rubella vaccine strain RA27/3 (75), attenuated poliomyelitis vaccine (mostly serotype 2 component) (76,77), and 17D yellow fever vaccine (78). On the other hand, if licensure of CVD 908 were to be pursued, sufficient empiric clinical data would have to be generated to document that the vaccinemias are not associated with any untoward reactions, as was done for licensure of attenuated rubella and oral polio vaccines. This would imply the need for large prelicensure safety trials. Accordingly, it was decided to pursue an alternative strategy whereby additional mutations were introduced into CVD 908 to yield a further derivative that would remain well tolerated and immunogenic yet would not manifest vaccinemias.

CVD 908-htrA Chatfield et al. (49) found that inactivation of htrA, a gene encoding a stress protein that functions as a serine protease, attenuated wild-type S. Typhimurium in the mouse model. Moreover, mice immunized orally with DhtrA S. Typhimurium were protected against subsequent challenge with a lethal dose of wild-type S. Typhimurium. Chatfield and coworkers thereupon introduced a deletion mutation into htrA of CVD 908, resulting in strain CVD 908-htrA (74) (Table 1). Tacket et al. (79) fed CVD 908-htrA as a single dose to three groups of subjects who ingested 5  107 (N ¼ 7), 5  108 (N ¼ 8), or 5  109 (N ¼ 7) CFU. The CVD 908-htrA strain was as well tolerated as the CVD 908 parent. Only one of 22 subjects developed a low-grade fever, which was detected by routine surveillance and was not associated with any complaints of malaise. However, 2 of the 22 subjects developed loose stools (79) (Table 2); mild diarrhea had not been observed in any recipients of CVD 908 (41,49). The immune response elicited by CVD 908-htrA was excellent: 20/22 individuals manifested significant rises in serum IgG O antibody, and in 100% of subjects, gut-derived IgA ACSs were detected that made antibody to O antigen (Table 2). These responses are virtually identical to what was observed in phase I clinical trials in subjects immunized with comparable doses of CVD 908. The one striking difference was with respect to vaccinemias. Whereas vaccinemias were detected in 12 of 18 subjects who received a 5  107 or 5  108 CFU dose of CVD 908, no vaccinemias were detected in any of the 22 individuals who ingested well-tolerated, highly immunogenic 5  107–9 CFU doses of CVD 908-htrA (p < 0.001). CVD 908-htrA was then evaluated in a phase II randomized placebo-controlled clinical trial in a larger number of adult North American subjects to assess the clinical acceptability and

immunogenicity of a lyophilized formulation of the vaccine administered following reconstitution (Table 2). Dosage levels of 5  107 and 5  108 CFU were evaluated (58). There were no differences in the rates of side effects among recipients of the high dose, the low dose, or placebo during 21 days of followup. The vaccine strain was immunogenic at both dosage levels, although responses were stronger in recipients of the higher dose (58,59).

CVD 909, a Derivative of CVD 908-htrA That Constitutively Expresses Vi Antigen Results of three large-scale field trials document that parenteral administration of non-denatured purified Vi polysaccharide vaccine stimulates serum Vi antibodies and confers a moderate level of protection against typhoid fever that lasts for two to three years (7,9,10,80). Results of other field trials show that attenuated strain Ty21a, which lacks Vi capsular polysaccharide, also confers a moderate level of longer-lived protection and achieves this by eliciting cell-mediated and humoral immune responses other than the stimulation of Vi antibodies. Levine (34) hypothesized that a live oral vaccine may achieve a much higher level of protection against typhoid fever if it stimulated anti-Vi antibody, in addition to stimulating antibody responses to other antigens and eliciting cell-mediated immunity. Regrettably, the new generation of attenuated S. Typhi vaccine strains, exemplified by strains such as CVD 908, CVD 908–htrA, Ty800, and M01ZH09, that are well tolerated and more immunogenic than Ty21a in eliciting serum O and H antibodies (51,60,62,79,81–83) only rarely stimulate serum Vi antibodies in subjects. Since Vi expression is highly regulated and appears to be turned off when S. Typhi gains its intracellular niche, Wang et al. engineered CVD 909 (84), a further derivative of CVD 908-htrA that constitutively expresses Vi (Table 1). CVD 909 was constructed by replacing the native promoter of tviA with the strong constitutive promoter Ptac. Constitutive expression of Vi was thereby achieved. In mice immunized mucosally (intranasally) with a single dose of vaccine, CVD 909 stimulated a significantly higher geometric mean titer (GMT) of serum Vi antibodies than CVD 908-htrA (84). In clinical trials, CVD 909 was as well tolerated as CVD 908-htrA and was comparably immunogenic in eliciting serum IgG O antibody and ASCs that make IgA O and H antibodies. CVD 909 did not consistently stimulate serum IgG anti-Vi antibody. However, CVD 909 was unique among the new generation of live oral typhoid vaccines in that at the higher dosages ingested (108–9 CFU), 16 of 20 subjects exhibited ASCs that made IgA anti-Vi (85); this demonstrated that CVD 909 elicited mucosal anti-Vi responses.

PBCC 211 and PBCC222 Deletions were introduced in aroA and aroD (encoding enzymes in the aromatic biosynthetic pathway) in wild-type strain CDC10-80 to produce vaccine candidate PBCC 211 (Table 1). Three different formulations of PBCC 211 were tested in phase I clinical trials (64). Among subjects who ingested either of two lyophilized formulations, a proportion developed fever. At the highest dosage levels tested, some vaccinees manifested silent self-limited vaccinemias on days 4 to 5 after ingesting a single dose of vaccine (64). A deletion in htrA was introduced into strain PBCC211 to derive vaccine strain PBCC222 (64). A lyophilized formulation of PBC222 contained in sachets was tested in phase I clinical trials in subjects who received dosage

78 11 24

Lyophilized

Freshly harvested

Freshly harvested

? 100%

? 100%

22 of 32 (69%) who ingested 108–9 CFU

3 (30%) 3 (33%)

17 (77%) 48 (62%) 10 (91%) 12 of 12 (100%) who ingested 108–9 CFU

Stool

Not done

Not done

0

0

~2 of 16 0 (13%) who ingested 109 CFU 0 0

0

0

0

0

0

0

0

>395

0

0

1 (4%)

1 (5%) 1 (1%) 0

>382

Fever

0

0

0

0

0

0

Blood

Positive cultures

a 16 of 20 recipients of a single 108–9 CFU dose of CVD 909 developed IgA Vi ASCs. Abbreviations: CFU, colony-forming units; ASCs, antibody-secreting cells.

Lyophilized

M01ZH09 (109)

16 (with bicarbonate buffer) 16 (without bicarbonate buffer)

48

Lyophilized

Lyophilized

9

Freshly harvested Frozen in glycerol

10

22

Number of subjects

Freshly harvested

Formulation

M01ZH09 (109)

X4073 (107–9) M01ZH09 (107–9) M01ZH09 (107–9)

CVD 908-htrA (107–9) CVD 908-htrA (107–8) Ty800 (107–10) CVD 909 (106–9)

Vaccine strain and dosage (CFU)

0

2 (13%)

4 (8%)

0

0

2 (9%) 5 (6.8%) 1 (9%) 2 (8%)

Diarrhea

11 of 15 (73%)

13/16 (81%)

20 (91%) 37 (47%) 6 (55%) 7/12 who ingested 108–9 CFU (58%) 9 (90%) 5 (56%) 8 of 16 (50%) who ingested 109 CFU

Serum IgG O antibody

14/15 (93%)

14/16 (88%)

9 (90%) 6 (67%) 23 of 32 (72%) who ingested 108–9 CFU

22 (100%) 68 (78%) 10 (91%) 5–6 per dosage (83–100%)a

IgA O ASCs

Immunologic responses

83

83

82

54

88

85

51

81

79

References

Table 2 Leading Live Oral Typhoid Vaccine Candidates Based on Results of Early (Phase I or II) Trials Assessing the Clinical, Immunologic, and Bacteriologic Responses of Adult Volunteers Following Ingestion of a Single Dose of Vaccine

Chapter 48: Attenuated Strains of S. Typhi and S. Paratyphi as Live Oral Vaccines 501

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Levine et al.

levels of 107 to 109 CFU (64). No vaccinemias were detected in recipients of this further derivative, but some who ingested the highest dosage level developed fever, chills, and headache, leading to abandonment of further clinical trials.

X3927, X4073, and X8110 Curtiss et al. demonstrated that cya (encoding adenylate cyclase) and crp (cyclic AMP receptor protein) constitute a global regulatory system in Salmonella that affects many genes and operons. They showed that a S. Typhimurium strain that harbors deletions in cya and crp (48) was attenuated compared with its wild-type parent and oral immunization–protected mice against challenge with virulent S. Typhimurium. Curtiss and team thereupon constructed vaccine candidate strain X3927 (Table 1), a cya,crp double mutant of S. Typhi strain Ty2, for use as a live oral typhoid vaccine and as a live vector (65,86). In phase I clinical trials, Tacket et al. (60) demonstrated that X3927 was attenuated compared with wild-type but insufficiently so to serve as a live oral vaccine in humans, since occasional subjects developed high fever and typhoid-like symptoms. Several subjects also manifested vaccinemias. In order to achieve a greater degree of attenuation, Kelly et al. (87) introduced into the cya, crp mutant a third deletion mutation in cdt, a gene that affects the dissemination of Salmonella from gut-associated lymphoid tissue to deeper organs of the reticuloendothelial system such as the liver, spleen, and bone marrow. The resultant cya,crp,cdt triple mutant strain, X4073 (Table 1), was fed to healthy adult North Americans, with buffer, in single doses containing 5  105, 5  106, 5  107, or 5  108 CFU. The strain was well tolerated except for one individual in the 5  106 CFU group who developed diarrhea (88). No subjects manifested vaccinemia. Four of five subjects who ingested 5  108 CFU exhibited significant rises in serum IgG O antibody and had ASCs that made IgA O antibody (88). The Curtiss group next introduced the cya,crp and cdt mutations into the modern Chilean wild-type strain ISP 1820, resulting in vaccine strain X8110 (Table 1), which was fed to volunteers at dosage levels of 105, 106, 107, and 108 CFU (89). No subjects developed fever, one of four who ingested the highest dosage level developed diarrhea, and two of eight who received 107–8-CFU manifested vaccinemia (89).

Ty445 and Ty800 Hohmann et al. constructed two candidate S. Typhi strains harboring deletions in phoP/phoQ (51,63). Strain Ty445 (Table 1), which also harbors a deletion in aroA, was found to be overly attenuated and only minimally immunogenic (63). In contrast, strain Ty800 (Table 1), a derivative of Ty2 deleted only in phoP/phoQ, was generally well tolerated and immunogenic when fed in dosage levels from 107 to 1010 CFU in a small phase I clinical trial involving 11 subjects (51) (Table 2). At the highest dosage level, 1 of 3 vaccinees developed diarrhea (10 loose stools). A double-blind, placebo-controlled phase II clinical trial of Ty800 at two dosage levels has been carried out in 183 healthy outpatient volunteers who received a single oral dose of 108 CFU (N ¼ 60), 109 CFU, or placebo (N ¼ 63). The vaccine was well tolerated compared with placebo, and a 4-fold increase in serum anti-LPS titers over predose level was observed in 36 of 55 recipients of 108 CFU (65.5%), and 44 of 55 who ingested 109 CFU (p < 0.001 versus placebo recipients) (ClinicalTrials.gov Identifier NCT00269295).

M01ZH09 Salmonella pathogenicity island 2 (SPI 2) encodes a type III secretion system that is necessary for S. Typhimurium to manifest full pathogenicity in the mouse model (90,91). S. Typhimurium strains harboring deletions in SPI2 do not manifest a full-blown systemic infection and show a diminished ability to replicate in macrophages. SPI2, which is activated under intracellular conditions, translocates effector proteins from the vacuole containing the Salmonella across the vacuolar membrane to the cytosol of the host cell (e.g., macrophages). ssaV forms part of the SSP2 secreton, the needle-like bacterial structure that transports proteins across the inner and outer bacterial membranes. S. enterica derivatives harboring mutations in ssaV are crippled in their ability to secrete SPI2 effector proteins. Deletion mutations in aroC and ssaV were introduced in wild-type strain S. Typhi strain Ty2 to derive vaccine candidate M01ZH09 (Table 1) (54). In a small phase I clinical trial that included nine adult subjects, M01ZH09 was well tolerated and elicited anti-Typhi immune responses in the majority of vaccinees (Table 2). The vaccine strain was recovered from stool cultures of three subjects. Several phase II dose-ranging clinical trials were subsequently carried out in the United States and in Vietnam to assess the clinical acceptability and immunogenicity of M01ZH09 and to evaluate formulations (Table 2) (82,83). Three groups of 16 healthy adults each ingested a single dose containing 5  107 CFU, 5  108 CFU, or 5  109 CFU in NaHCO3 buffer, while 12 subjects received placebo in a randomized, doubleblind trial. The vaccine was well tolerated at all dosage levels, with only a few mild febrile responses. Bacteremia was not detected in any vaccine, and fecal shedding was abbreviated (82). All recipients of the 5  109 CFU dosage exhibited IgA anti-LPS ASCs, and 50% manifested rises in serum IgG anti-LPS antibody. In another phase II trial in U.S. adults, 32 subjects were randomly allocated to receive as single 5  109 CFU dose of M01ZH09 with or without NaHCO3 buffer (that also contained ascorbic acid and aspartame) to neutralize gastric acid (83). The vaccine was well tolerated, although two subjects who received vaccine with buffer experienced diarrhea. Fecal shedding was abbreviated (seven days or less). Surprisingly, there was no difference in the rate or magnitude of immune responses between these groups (Table 2); 14 of 16 recipients of vaccine with buffer (88%) and 14 of 15 individuals who ingested vaccine without buffer (93%) exhibited rises in ASCs making IgA anti-LPS antibodies. Similarly, 4-fold rises in serum IgG anti-LPS antibodies were observed in 13 of 16 (81%) of vaccinees who got vaccine with buffer and in 11 of 15 (73%) who received vaccine without buffer. The clinical trials in U.S. adults were followed by a phase II trial in 27 adults in Vietnam, which showed the vaccine to be as well tolerated and comparably immunogenic in that population as in U.S. adults. This was followed by a phase II study in Vietnamese children 5 to 14 years of age who received a single dose of vaccine (N ¼ 101) or placebo (N ¼ 50). The vaccine was again reported to be well tolerated and immunogenic in the children.

Salmonella Paratyphi A Strains CVD 1902 and CVD 1903 Given the ability of attenuated strains of S. Typhi to function as live oral vaccines that prevent typhoid fever and in view of the partial protection in some field studies conferred by oral Ty21a

Chapter 48: Attenuated Strains of S. Typhi and S. Paratyphi as Live Oral Vaccines

against S. Paratyphi B disease (2,3,92), one rational strategy to prevent S. Paratyphi A and B disease is by the development of attenuated strains of these serovars to serve as live oral vaccines. Accordingly, Vindurampulle et al. (93) engineered two candidate live oral S. Paratyphi A vaccine strains. On the basis of the excellent preclinical and clinical experience with guaBA mutants of S. Typhi (47) and Shigella (94,95), a deletion in guaBA was introduced into wild-type S. Paratyphi as the primary attenuating mutation. Additional deletion mutations were then made in either clpX or clpP to derive candidate vaccine strains CVD 1902 and 1903, respectively. Each of these strains was markedly attenuated compared with the wild-type parent. Preparations are in progress to initiate a phase I clinical trial.

FUTURE USE OF LIVE ORAL TYPHOID VACCINES An ideal attenuated strain constituting the live oral vaccine of the future will be so well tolerated that it will be possible to administer the vaccine routinely to infants as well as to immunocompromised subjects with AIDS or other immune deficiencies. On the other hand, the ideal live vaccine will be so immunogenic that a single dose will confer a high level of long-term protection that will endure throughout childhood, including during the usual peak risk for typhoid fever during the period 5 to 19 years of age. With such properties, it will be possible to pursue control of typhoid by a strategy involving routine immunization of infants within the expanded program on immunization schedule supplemented by school-based immunization campaigns. An alternative epidemiologic approach to control endemic typhoid fever would be to institute only school-based immunization programs. Since peak incidence of typhoid fever in most endemic areas occurs in school-age children 5 to 19 years of age, and since this is a ‘‘captive’’ population, it should be possible in the future to design control programs to incorporate school-based immunization with a single-dose oral vaccine. Field experiences with Ty21a support such an approach. Even using multiple-dose regimens of Ty21a, Ferreccio et al. (33) reported the practicality of school-based immunization in a field trial in 216,692 schoolchildren that compared two-dose, three-dose, and four-dose regimens (all within eight days) of Ty21a. Moreover, Levine et al. (34) observed a herd immunity effect in geographically separate areas of metropolitan Santiago when large-scale use of Ty21a was carried out in the course of field trials in other areas of the city. Practicality will be greatly enhanced with the advent of a single-dose live oral vaccine, and herd immunity effects should be even more pronounced with a vaccine that exhibits greater efficacy than Ty21a.

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49 Oral Cholera Vaccines Jan Holmgren University of Gothenburg Vaccine Research Institute (GUVAX) and Department of Microbiology & Immunology, University of Gothenburg, Gothenburg, Sweden

James B. Kaper Department of Microbiology and Immunology, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION Cholera remains an important global health challenge. It is the most severe of all diarrheal diseases and the archetype for the large group of infectious diarrheas caused by noninvasive bacteria that produce one or more enterotoxins. Collectively, these ‘‘enterotoxic enteropathies’’ (1) account for a substantial proportion of all infection-related diarrheas in the world. Although not as prevalent as enterotoxigenic Escherichia coli (ETEC), cholera is notable for the striking rapidity with which it can lead to severe dehydration, shock, and death after onset of symptoms and for causing explosive outbreaks. Although accurate numbers are difficult to obtain, in part because of surveillance difficulties but also for fear of economic and social consequences, cholera is estimated to account for at least 3 to 5 million cases and 120,000 to 200,000 deaths annually among adults as well children (2). Although a parenteral killed whole-cell (WC) cholera vaccine is still available, its use has not been recommended by WHO since the late 1970s. The protective efficacy was generally modest and of short duration (<50% for <6 months), local side effects made the vaccine poorly accepted, and it did not prevent transmission of the infective agent (3,4). Despite reports of new parenteral subunit vaccine preparations, only one, a Vibrio cholerae O1 Inaba polysaccharide-cholera toxin conjugate, has been tested in humans (5). This preparation engendered vibriocidal antibodies but has not been tested for protection against experimental challenge or natural disease. Over the last 25 years, most work on new cholera vaccines has been to develop oral vaccines by one of two broad approaches: (i) the use of killed WC V. cholerae bacteria with or without purified cholera toxin B (CTB) subunit and (ii) attenuated recombinant strains used as live oral vaccines. Whereas both of these approaches have yielded licensed vaccines, only an oral killed vaccine (Dukoral1, Crucell-SBL Vaccines, Stockholm, Sweden) is commercially available at this writing. Oral cholera vaccines, both killed and living, will be reviewed in this chapter.

V. CHOLERAE AND CHOLERA PATHOGENESIS AND DISEASE The Pathogen and Its Epidemiology V. cholerae of serogroup O1 is the causative agent of at least 98% all cholera cases in the world (a small percentage in Southeast Asia is caused by V. cholerae O139). V. cholerae O1 can appear as

one of two serotypes, Inaba and Ogawa, and be of either the classical or El Tor biotype, the latter predominant since the 1970s (2). A feared characteristic of V. cholerae distinguishing it from most other enteric pathogens is the propensity for causing large epidemic outbreaks and even pandemics, which historically have affected large parts of the world (6). The latest, still ongoing, seventh pandemic originated in the Celebes in 1961 and since then has spread to and become endemic in many countries in Asia and Africa. In 1991, the seventh pandemic, caused by the El Tor biotype, spread to most of South and Central America, bringing cholera back to this geographic area for the first time in more than 100 years. When an epidemic strikes an area where sanitation and hygiene are poor and health care is inadequate, the results can be disastrous. This was illustrated during the refugee crisis in Goma, Zaire in 1994, when an estimated 58,000 to 80,000 cases and 23,800 deaths occurred within one month (7). Although in endemic areas the highest incidence of cholera is seen in children below five years, with annual cholera incidence rates exceeding 10 cases per 1000 children in some areas, approximately two-thirds of all V. cholerae O1 cases occur in older children and adults. When cholera spreads to new regions and populations, all age groups are affected to the same degree. This pattern is probably explained by natural immunity that develops with age in endemic countries; this background immunity is lacking in newly infected populations (2,8,9). In 1993, a new V. cholerae serogroup, O139 Bengal, appeared in Bangladesh and eastern India and caused disease in all age groups, indicating that age-related immunity acquired to the O1 serogroup did not extend to O139 infection. The rapid emergence of this serogroup accompanied by high attack rates, initially suggested that it might lead to an 8th pandemic. However, the incidence of O139 disease quickly fell to below 5% of all cholera cases (2), and infection was only seen in a few countries in Southeast Asia.

Cholera Disease The disease caused by toxigenic V. cholerae O1 and O139 is characterized by watery diarrhea without blood and mucus (2). The acute, profuse watery diarrhea usually lasts for a few days. In a proportion of cholera cases, however, life-threatening dehydrating disease and acidosis ensue. The case fatality rate

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in severe, nontreated cholera is 30% to 50%. Persons of blood group O have an increased risk of developing severe cholera (cholera gravis) when infected (10–14). The keystones to treatment of cholera gravis are aggressive rehydration therapy and antibiotics.

Virulence Factors and Pathogenesis Cholera is primarily a disease originating from the upper part of the small intestine. Intestinal perfusion studies have revealed that as much as 90% of the intestinal secretion occurs in the uppermost 1 m of the intestine in adult cholera patients. V. cholerae O1 (and O139) bacteria have developed special, highly efficient means to colonize and multiply to prodigious numbers in the small intestine, and in this process they also efficiently produce and release cholera toxin (CT) (2,15). Through its high-affinity binding to the gut epithelium and its subsequent cellular action, CT is directly responsible for the pathogenic effects on intestinal ion and water secretion processes that may lead to life-threatening diarrhea and dehydration (16,17). The most important attributes of V. cholerae allowing it to efficiently colonize the small intestine include (i) the toxin-coregulated pilus (TCP), which has been found to be a critical attachment fimbriae in at least the early stage of colonization (18,19); (ii) a soluble Zn-metalloprotease, hemagglutinin/protease (HapA) that can degrade mucin and epithelial tight junction-associated proteins and facilitates bacterial penetration through the intestinal mucus layer (20); (iii) and the single flagellum, which operates in concert with chemotactic receptors and intracellular sensor molecules to allow directed motility toward the intestinal cell wall (21). Expression of virulence factors is controlled by complex regulatory systems that include factors such as ToxT/ToxR regulator proteins, quorum sensing, small RNA molecules, and marked differences between in vivo and in vitro expression (22). The identification of the subunit structure and function of CT was of pivotal importance for clarifying the pathogenesis of cholera (reviewed in Refs. 16,17). The 84 kDa CT consists of five identical B subunits (11.6 kDa) forming a ring structure into which a single A subunit (28 kDa) is noncovalently inserted. The CTB pentamer attaches CT to the intestinal epithelial cell through its high-affinity binding to cell-surface receptors, the monosialoganglioside GM1. Production of a neuraminidase by V. cholerae increases the number of GM1 receptors on host cells. After binding to GM1, which appears to be localized mainly in lipid rafts on the cell surface, CT is endocytosed by the cell. After endocytosis, CT travels to the endoplasmic reticulum (ER) via a retrograde transport pathway, possibly via the Golgi-system. After CT has reached the ER, CTA or CTA1 dissociates from CTB to enter the cytosol, where the CTA1 polypeptide then catalyzes the ADP ribosylation of the trimeric Gsa component of adenylate cyclase (AC). This locks AC in its GTP-bound state, resulting in enhanced AC activity and increased cAMP production. The resulting higher levels of intracellular cAMP causes an imbalance in electrolyte transport across the epithelial cell, with a decrease in sodium uptake and an increase in chloride and bicarbonate export. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, which is activated by cAMP-responsive protein kinases, is the major factor in the marked efflux of chloride ions. Water follows this imbalanced ion gradient to produce a massive net fluid loss from the intestine that will soon lead to dehydration. In addition to the direct effect of CT on AC activity and cAMP production in the enterocytes,

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studies in experimental animal studies indicate that the diarrheal response to CT might have a significant neurological component, involving stimulation by CT of serotonin release from intestinal enterochromaffin cells and serotonin-induced release of vasointestinal peptide (VIP) from local neural networks, leading to fluid secretion through VIP-induced increase of cAMP production in enterocytes (23). TCP, a type 4 pilus that is closely related to other enterobacterial type 4 pili, has been shown to be essential for colonization and virulence in humans (19). The detailed mechanisms by which TCP promotes colonization have remained elusive. It has been proposed (24) that the initial attachment of bacteria to the epithelium is mediated by an outer membrane protein rather than by TCP, and that TCP together with a soluble protein (TcpF), secreted via the TCP biogenesis apparatus, mediate bacterial microcolony and biofilm formation on the epithelial surface as critical events in the colonization process (25). Although CT and TCP are undoubtedly the main virulence factors of V. cholerae, several accessory factors have also been described (reviewed in Ref. 15). In addition to HapA and neuraminidase, additional soluble factors include a variety of ‘‘minor toxins’’ that might contribute to cholera diarrhea. Among these are (i) Zot and Ace, which have enterotoxic activity and are part of the CTXF phage that encodes CT; (ii) RTX toxin, an actin-cross-linking toxin produced by El Tor but not classical strains; (iii) S-CEP, a cytotonic protein that elongates Chinese hamster cells; and (iv) hemolysin, a poreforming and vacuolating toxin. Even though the role of all of these factors in the virulence of V. cholerae is probably minor relative to CT and TCP, they have been suggested to contribute, singly or jointly, to mild diarrhea and other symptoms induced by live attenuated vaccines when fed to volunteers (see the following text).

IMMUNE MECHANISMS IN CHOLERA Innate Immunity Susceptibility to infection with V. cholerae is dependent on both adaptive immune responses induced by previous infection or vaccination and on innate host factors. In contrast to the adaptive immune mechanisms, which have been studied extensively in cholera, little is known about the innate immune components and mechanisms. Among the intrinsic host factors that influence susceptibility to cholera, stomach acidity and ABO blood groups are the most studied. In Bangladesh, patients with cholera (or ETEC diarrhea) have significantly lower gastric acid levels than other groups studied, and low gastric acid level is also associated with more severe cholera disease (26). It is also well known that neutralization of stomach acid dramatically reduces the minimal infectious dose of V. cholerae (2,17). In healthy North American volunteers given different doses of V. cholerae O1 bacteria, the average minimal pathogenic dose was 106 to 108 when given without bicarbonate but 1000- to 10,000-fold less when the bacteria were given together with 2 g of sodium bicarbonate. It has been calculated that in endemic situations, the average infectious dose of cholera vibrios when ingested together with rice or other food, resulting in transient neutralization of stomach acidity, is approximately 103 organisms. Likewise, it is well established that people who have undergone surgical removal of the acid-producing part of the stomach have an increased risk of cholera infection and disease.

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Several case-control studies found that individuals with blood group O are at increased risk of hospitalization because of both V. cholerae O1 and V. cholerae O139. In a prospective study of household contacts of (O1 El Tor) cholera patients in Bangladesh, Glass et al. (11) reported that contacts with blood group O had 5 to 10 times increased risk of getting moderate or severe cholera compared with contacts with blood group A or B, and a more than 20-fold increased risk compared with blood group AB contacts. In a later study from the same area performed between 1985 to 1987, at which time cholera was caused at about the same frequency by V. cholerae O1 of El Tor and the classical biotypes, Clemens et al. (12) found that the link between blood group O and cholera severity appeared to be restricted to El Tor cholera and was not seen for classical biotype cholera. Increased severity of disease was also seen in North American volunteers with blood group O who were challenged with V. cholerae O1 of either classical or El Tor biotypes (10,13). Harris et al. (14) proposed that adaptive immune responses from prior infection, not fully measured by serum vibriocidal titers, also influence the correlation between blood group O and severity of disease. Many factors in addition to gastric acidity and ABO blood group may contribute to the innate defense against cholera. A whole-genome microarray screening was recently used to study gene expression in duodenal mucosal biopsies from patients with acute cholera (27). Among about 21,000 genes expressed in the intestinal epithelium, the majority of 29 early upregulated genes have a described role in the innate defense against infections. These include a number of proteins with antibacterial activity plus a set of genes that appear to have been indirectly activated by CT through CT-induced activation of IL-1.

Adaptive Immune Mechanisms The best-studied correlate of adaptive immunity to V. cholerae is serum vibriocidal antibody titer. Seroepidemiologic studies have shown that in cholera-endemic areas, vibriocidal antibodies increase with age and that the risk of disease is inversely proportional to the vibriocidal antibody titer (8,28). However, vibriocidal antibodies in both unvaccinated and vaccinated individuals are only a surrogate marker for the intestinal mucosal immune status. For instance, parenteral vaccines confer only limited and short-lived protection even though they induce extremely high vibriocidal antibody titers (4). Instead, available evidence indicates that immune protection in cholera, both that mediating recovery from ongoing infection and disease and that preventing cholera infection and disease after effective immunization, depends on the stimulation of a local-mucosal immune response in the intestine. The main findings, which have also directly guided the development of more effective cholera vaccines, can be summarized as follows:

1.

2.

In animal models, antibacterial and antitoxic antibodies capable of preventing bacterial colonization and the binding and action of CT in the small intestine were found to effectively protect against experimentally induced V. cholerae infection and disease. It was further noted that in the intestine, such antibacterial and antitoxic antibodies produce a synergistic cooperative effect in protection against disease. Both antibacterial and antitoxic immunity were found to depend mainly, if not exclusively, on locally produced mucosal antibodies of the secretory immunoglobulin IgA

3.

(S-IgA) type directed mainly against lipopolysaccharide (LPS) and CTB, respectively. Stimulation of a protective gut mucosal immune response was accomplished much more efficiently by oral immunization than by parenteral immunization.

Antitoxic Cholera Immunity Studies in experimental animals showed a direct correlation between protection against CT-induced fluid secretion and intestinal synthesis of SIgA antibodies (29), and also between protection and the number of SIgA antitoxin-producing cells in the intestine (30). A protective role of SIgA antitoxin was also indicated by the direct correlation in breast-fed children in Bangladesh of a reduced risk of developing disease after infection with V. cholerae O1 and the ingestion of mother’s milk having SIgA antitoxin antibodies above a certain level (31). Furthermore, cholera vaccine-induced antitoxic immunity associated with intestinal SIgA antitoxin production was shown to confer significant, although short-lived, cross-protection against diarrhea caused by LT producing ETEC in a large field trial in Bangladesh (32). The identification of the subunit structure of CT and the roles of the different subunits in pathogenesis and immunity suggested that the isolated CTB subunit moiety should be a useful vaccine component for inducing protective antitoxic immunity. This notion was strengthened by findings in animals showing that immunization with highly purified CTB gave rise to toxin-neutralizing antibodies, which could fully protect against disease also after challenge with live cholera bacteria in the gut (33). Furthermore, CTB was found to be particularly well suited as an oral immunogen, as it is stable in the intestinal milieu and capable of binding to the intestinal epithelium, including the M cells of the Peyer’s patches, properties that are critically important for stimulating mucosal immunity and local immunological memory.

Colonization Factors and Antibacterial Immunity It is well established that V. cholerae O1 LPS is the predominant antigen inducing protective antibacterial immunity against experimental cholera caused by O1 bacteria (34,35). Immunologically, the O1 LPS contains group-specific epitopes shared between the Inaba and Ogawa serotypes and additional serotype-specific epitopes. Studies have shown that both antibodies against the main shared epitope(s) and serotype-specific antibodies can protect against experimental V. cholerae O1 infection (34). Thus, it may be advantageous but not absolutely critical for a cholera vaccine to contain both Inaba and Ogawa LPS to protect against both serotypes of V. cholerae. Protective immunity against V. cholerae O139 also appears to be mediated predominantly by antibodies to (O139) LPS (36). When human convalescent sera with high vibriocidal titers to V. cholerae O1 are absorbed with O1 LPS, approximately 80% of the killing activity is removed (37). The identity of the antigen(s) responsible for the remaining vibriocidal activity is unknown but appears to be proteinaceous in nature. One antigen for which importance in human disease has been well established is the TCP. V. cholerae O1 and O139 strains specifically mutated in the tcpA gene encoding the major pilin subunit do not colonize or induce diarrhea in volunteers (19,38). Transcutaneous immunization with TcpA induces protective immunity against V. cholerae O1 challenge in a mouse model

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Table 1 Protection Against Cholera by the Oral B Subunit-Killed Whole Cell (Cholera Toxin B-Whole-Cell) and Whole Cell–Only Vaccines in the Bangladesh Field Trial Protective efficacy percentage (95% CI) Follow-up period 4–6 mo 1st yr 2nd yr 3rd yr

Cholera toxin B-whole-cell vaccine All ages 85% 64% 52% 19%

(56–95%) (50–74%) (30–76%) (Nil–46%)

2–5 yr

>5 yr

100% 38% 47% Nil

76% 78% 63% 41%

Whole-cell-only vaccine All ages 58% 56% 55% 41%

(14–79%) (39–76%) (33–69%) (7–62%)

2–5 yr

>5 yr

35% 31% 24% 2%

71% 67% 73% 61%

Source: From Refs. 44 and 45.

(39). However, little, if any, anti-TCP immune response was seen in North American volunteers infected with wild-type V. cholerae O1 (19), although it was reported that the majority of Bangladeshi patients with natural cholera infection developed both a mucosal and a systemic IgA response to the TcpA subunit (40). It remains to be determined whether mucosal immune responses against TCP and other surface antigens on V. cholerae could add significantly to the protective action mediated by antibodies to O1 (or O139) LPS antigen in humans. An important observation guiding the design of oral cholera vaccines, especially the oral WC-CTB cholera vaccine, is the synergistic cooperation between antitoxic and antibacterial immune mechanisms in mediating protection. Either of the two main protective antibodies (directed against the LPS and CTB) can independently contribute to protection against disease by inhibiting bacterial colonization and toxin binding, respectively (33,34,41). When present together in the gut, these antibodies have been shown to have a strongly synergistic protective effect (33,41).

KILLED WHOLE-CELL CHOLERA VACCINES Killed WC-rCTB Vaccine The only commercially available oral cholera vaccine at present is the WC-rCTB vaccine (DukoralTM), consisting of killed WC V. cholerae O1 bacteria (classical and El Tor biotypes, Inaba and Ogawa serotypes) in combination with recombinant (42) B-subunit of CT. The vaccine is licensed in more than 50 countries worldwide and recommended and prequalified by WHO for UN agency purchasing (3,43). After having been given in more than 10 million doses together with a bicarbonate buffer to people in countries with good systems for registration of adverse reactions, the post-licensure conclusion is that this vaccine is exceptionally safe and well tolerated. The only adverse effect reported is occasional mild gastrointestinal disturbances in persons sensitive to the bicarbonate buffer. The WC-rCTB vaccine has also been found to be well tolerated by HIV-positive individuals, by pregnant or breast-feeding women, and by children and infants as young as six months. In a large randomized field trial in Bangladesh in the late 1980s, a three-dose regimen of WC-CTB vaccine was tested compared with either WC vaccine alone or placebo (killed E. coli K12 bacteria) (44,45). At this time the CTB antigen was not made from recombinant CTB-only producing strains but was prepared and extensively purified after first isolating CT on a GM1-affinity column and then separating the A and B subunits by repeated chromatographic steps (46). In comparison with the placebo group, the WC-CTB vaccine gave 85% and 50% cholera-specific protection when assessed after four to six months (44) and three years (45), respectively, in all age groups

and two doses conferred similar efficacy as three doses. In children older than five years and in adults, protective efficacy remained high for the first two years of follow-up and was evident also during the third year (Table 1). In children two to five years of age, on the other hand, the 100% efficacy noted during the first four to six months of surveillance (44) waned more rapidly to be ca. 40% during the first two years of followup, and not detectable during the third year after vaccination (45) (Table 1). In a placebo-controlled phase III trial in Peru in 1993 in military recruits (47), two doses of the WC-rCTB vaccine given one to two weeks apart induced 86% protection in the vaccinees against an outbreak of cholera occurring ca. three to five months after vaccination, that is, very similar to the 85% protection after four to six months noted in the previous Bangladesh trial. Importantly, this high level of vaccineinduced protection was achieved against cholera due to V. cholerae O1 El Tor in a previously unexposed population. In a later study in Peru (48), two doses of the vaccine did not appear to give significant protection during the first year of surveillance, but the study was criticized for lack of rigor during this observation period (49). In the second year of follow-up, after a booster dose had also been given the efficacy was 82% (48). The O1 WC/rCTB vaccine was also tested with very good results in a large effectiveness trial in Mozambique (50). The vacccine was administered in a two-dose regimen through the normal public health system to a high-endemicity population with a high seroprevalence of HIV infection (estimated to be ca. 30% in women of child-bearing age). Despite the potential immunocomprising impact of HIV infection, the protection was found to be high, ca. 80% against all hospital admissions for cholera and ca. 90% against those hospital admissions that were associated with severe dehydration, thus confirming the previous phase III efficacy trial findings in both Asia and Latin America. Through its CTB component the WC-rCTB vaccine also has been shown to give 50% to 70% protection against diarrhea (and as much as 86% protection against life-threatening disease) caused by LT-producing ETEC (32,51,52) (see Svennerholm et al., this volume).

Killed WC-Only Vaccines When tested side-by-side in Bangladesh with the WC-CTB vaccine, a vaccine (WC) containing the identical WCs but no CTB provided significant short-term as well as long-term protection against cholera (Table 1) (44,45). The short-term protection, 58% for the initial four- to six-month period, was however significantly lower than the 85% efficacy of the combined WC-CTB vaccine during the same period, which

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indicates an independent protective immunogenic effect of the CTB component in the latter vaccine. Indeed, if one compares the rate of cholera in the WC-CTB group with that in the WC group, the added protective efficacy owing to the addition of CTB to WC was 73% during this period (44). The WC-CTB vaccine continued to be significantly more protective than the WC-alone vaccine for the first 9 months after vaccination (45). Thereafter, the overall efficacy was similar, approximately 55% during the second year of follow-up and 20% to 40% during the third year. A killed V. cholerae O1 WC vaccine, modeled on the Swedish WC-rCTB vaccine but lacking the B-subunit, has been locally produced, tested, and licensed in Vietnam. Immunization by two oral doses of this vaccine resulted in 66% protective efficacy during a local cholera outbreak that occurred 8 to 10 months after vaccination (53). Notably, when there was a cholera outbreak three or five years after the locally produced vaccine had been given in a two-dose schedule under public health conditions, there was a ca. 50% protection compared with unvaccinated individuals (54). A second-generation bivalent vaccine (O1/O139-WC), containing killed vibrios of the serogroup O139 in addition to a slightly altered composition of O1 strains (now made identical to the Swedish vaccine) has also recently been developed in Vietnam (55). Phase II studies with this vaccine indicate that it is safe and immunogenic, can be administered without buffer, and elicits antibacterial immune responses in both adults and children. The vibriocidal antibody responses to O1 V. cholerae were similar to those obtained with the international O1 WC-rCTB vaccine. With support from the International Vaccine Institute (IVI), South-South technology transfer from Vietnam to producers in India and Indonesia has been initiated to facilitate local production and introduction of the O1/O139 WC oral cholera vaccine in developing countries that have endemic cholera. A large phase III placebo-controlled, randomized trial is being conducted in Kolkata, India, to assess the protective efficacy induced by this vaccine.

Herd Protection and Overall Reduction of Diarrhea Morbidity and Mortality A recent reanalysis of indirect and direct effects of vaccination with killed oral cholera WC-CTB and WC vaccines in the large field trial in Bangladesh indicated the presence of substantial herd protection effect (56). An inverse relationship between the level of vaccine coverage in a residential cluster (a ‘‘bari’’) and the incidence of cholera in individual placebo recipients residing in the bari was found. Vaccine (including placebo) coverage of the population targeted for enrollment in the trial ranged from 4% to 65%. Incidence rates of cholera among placebo recipients were inversely related to levels of vaccine coverage (7.01 cases/1000 in the lowest quintile of vaccine coverage vs. 1.47 cases/1000 in the highest quintile, corresponding to a 79% indirect protection for the placebo recipients in the baris with the highest coverage; p < 0.0001 for trend). In contrast, in the quintile with the highest level of vaccine coverage (>51%) direct protection was only 14%, the lowest of any quintile. After adjustment for the level of vaccine coverage of the cluster, specific vaccine direct protective efficacy overall remained significant (55%, p < 0.0001). These results indicate that in addition to providing direct protection to vaccine recipients, killed oral cholera vaccines confer significant herd protection to neighboring non-vaccinated individuals. Use of these vaccines could have a major effect on the burden of cholera in endemic

Figure 1 Estimated combined impact of specific efficacy and indirect herd protection of oral cholera vaccines in control of cholera in endemic settings at different vaccination coverage. Source: Adapted from Ref. 57.

settings. Indeed, the latter conclusion was further emphasized by mathematical modeling of the calculated effect of public health use of killed cholera vaccines in a setting such as that in Bangladesh (57). The results indicate that through the combination of direct (vaccine-specific) and indirect (herd protection) protection, repeated routine immunization with oral cholera vaccine could practically eliminate cholera in high-endemic settings and thus be an important tool in the public health control of cholera. Thus, recent estimates indicate that already a vaccination coverage of 50% could result in more than 90% reduction of cholera in an endemic area because of the combined impact of direct efficacy and herd protection (Fig. 1) (57). Consistent with the demonstrated efficacy of oral WC-CTB and WC-only cholera vaccines and the importance of cholera and ETEC as causes of severe watery diarrhoea in Bangladesh, both vaccines were found to substantially reduce the overall diarrhea morbidity (58). Admissions for severe watery diarrhea were significantly reduced, by 51% and 32%, in the WC-CTB and WC vaccinated groups, compared with the placebo group during the first year after vaccination in the Bangladesh trial. In the first year of follow-up after vaccination, there was also a dramatic effect of cholera vaccines on total mortality (58). Thus, compared with placebo, overall mortality rates were 26% lower in the WCCTB group and 23% lower in the WC group during the first year.

Killed O139 Cholera Vaccine Candidates Both killed bivalent O1/O139 WC-rCTB and O1/O139 WConly vaccines modeled on the licensed O1 WC-rCTB vaccine were developed in Sweden and found to be safe and immunogenic in volunteers (59). However, in expanded phase II studies vibriocidal responses against the O139 component were less frequent and at lower titers than against V. cholerae O1 (P. Askelo¨f, personal communication). When the O1/O139 WCrCTB vaccine was tested in North American volunteers, the protective efficacy against challenge with a virulent O139 strain was also less than that against V. cholerae O1 El Tor Inaba challenge (D.A. Sack et al., unpublished).

LIVE ATTENUATED CHOLERA VACCINES A number of different live attenuated cholera vaccines have been developed and undergone clinical trials. The crucial

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mutation in these strains is the deletion of gene sequences encoding the CT A-subunit (ctxA), which is responsible for the ADP-ribosylating activity of the holotoxin. Beyond this basic mutation, the various vaccine candidates differ in other mutations incorporated into the final strain or the parent wild-type V. cholerae used as starting material.

Early Generations of Recombinant Vaccine Candidates The first generation recombinant cholera vaccines were generated from wild-type El Tor strain N16961 and classical strain 395. These vaccine candidates, strain JBK70 from N16961 and strains CVD 101 and 395N1 from 395, were markedly attenuated compared with the wild parent strain and were highly immunogenic (19,60). When volunteers who were immunized with a single dose of 106 JBK70 were challenged with the virulent parent strain, significant protection was observed (60). Diarrhea occurred in 7 of 8 nonimmunized controls but in only 1 of 10 vaccinees, a vaccine efficacy of 89%. This level of efficacy was equivalent to that seen following sequential experimental infections with wild-type El Tor strains. Interestingly, JBK70 produced neither the A nor B subunits of CT; so this challenge study demonstrated the importance of antibacterial immunity in the absence of antitoxic immunity. Despite the high levels of immunity engendered by these three vaccine candidates, they were unexpectedly reactogenic. Approximately, one half of the recipients of JBK70, CVD 101, and 395N1 suffered adverse reactions such as mild diarrhea, malaise, nausea, vomiting, abdominal cramps, low-grade fever, headache, and increased intestinal inflammation (19,60,61). These strains never caused severe or even moderate diarrhea but were nonetheless not studied further because of these reactions. These results were surprising since it was previously believed that CT was the only diarrheagenic factor produced by V. cholerae. Two hypotheses were proposed to explain this response (60). The first hypothesis was that a previously unknown enterotoxin was responsible for the diarrhea in the absence of CT. Two new toxins, Zot (Zonula occludens toxin) and Ace (Accessory cholera enterotoxin), were discovered whose genes were adjacent to the ctx genes (62,63). However, when the zot and ace genes were deleted along with the ctxA gene, the resulting vaccine candidate, CVD 110, still was unacceptably reactogenic (64). The second hypothesis was that avid colonization by the attenuated V. cholerae strain in the proximal small bowel, a site where only low numbers of bacteria are found in healthy North Americans, would somehow disturb the normal balance of secretion and absorption resulting in diarrhea and other symptoms. A vaccine candidate derived from a wild-type strain that did not colonize as avidly as N16961 or 395, CVD 103-HgR, was well tolerated and immunogenic in volunteers (see below). Although the exact reason(s) for the reactogenicity of these early vaccine candidates is not known with certainty, recent evidence suggests that the V. cholerae flagellin protein, the major subunit of flagella, can induce intestinal inflammation via activation of TLR5 and production of IL-8 (65).

V. cholerae CVD 103-HgR The first recombinant V. cholerae vaccine strain to be well tolerated yet highly immunogenic and protective was strain CVD 103, derived from the classical Inaba V. cholerae O1 strain

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569B. This parent strain colonized the intestine at lower levels than other toxigenic V. cholerae strains and was reported to lack a Shiga-like toxin activity that was possibly involved in vaccine reactogenicity. CVD 103 was derived from 569B by deletion of the ctxA subunit (66) and a further derivative was constructed by inserting genes encoding resistance to mercury (mer) into the chromosomal hlyA locus. The mercury resistance provides a marker to readily differentiate the vaccine strain from wild-type V. cholerae. The resulting strain, CVD 103-HgR, exhibits many of the characteristics of an ideal cholera vaccine and was the first recombinant bacterial vaccine to be licensed for human use. CVD 103-HgR has been extensively studied in multiple randomized, placebo-controlled, double-blind phase I and II clinical trials involving more than 7000 subjects in countries in Asia, Latin America, Africa, Europe, and North America (reviewed in Refs. 17,67–69). The safety and immunogenicity of this vaccine has been demonstrated in subjects as young as 3 months and as old as 65 years, including subjects infected with the human immunodeficiency virus (HIV) (70,71). In all studies, neither diarrhea nor any other adverse reaction occurred significantly more often in vaccinees than in placebo recipients. Multiple efficacy studies were conducted, in which North American volunteers were vaccinated with CVD 103HgR and then challenged along with unvaccinated controls with virulent toxigenic V. cholerae O1 strains. A single dose of CVD 103-HgR provided significant protection against challenge with classical Inaba, classical Ogawa, El Tor Inaba, or El Tor Ogawa strains (72–74). In a multicenter, randomized, doubleblind, placebo-controlled efficacy trial of a single dose of classical CVD 103-HgR against challenge with virulent El Tor strain N16961, moderate or severe diarrhea (>3 L) was seen in 9 of 23 placebo recipients versus 1 of 28 vaccinees (91% efficacy). When diarrhea of any severity was assessed, 21 of 23 placebo recipients and 5 of 28 vaccinees had any diarrhea (mild to severe) (80% efficacy) (73). Another trial demonstrated that protection is evident as early as eight days after vaccination and lasts for at least six months (the shortest and longest intervals tested) (75). The single-dose efficacy and rapid onset of protection are attractive advantages for vaccination in an outbreak situation. On the basis of the demonstrated safety, immunogenicity in many populations and efficacy in multiple experimental challenge studies in volunteers, CVD 103-HgR was licensed in a number of countries where it was used mainly for prevention of cholera among travelers to high-risk areas where cholera was endemic or epidemic. Although the protective immunity engendered by CVD 103-HgR in North Americans was well established in volunteer challenge studies, the record of protective immunity in choleraendemic countries is mixed. Results from a large randomized, double-blind, placebo-controlled field trial involving 67,508 pediatric and adult subjects in Indonesia (76) who were randomized at the level of individual subject showed that a single dose of vaccine did not confer significant long-term protection over the four-year observation period (13.5% vaccine efficacy overall). Unfortunately, too few cases occurred in the first six months of follow-up to definitively assess whether CVD 103HgR provided short-term protection similar to the short-term duration of North American clinical trials volunteers but a post-facto analysis suggested a protective efficacy of 60% (M. Levine, personal communication). It is of interest that in a population where the incidence of El Tor cholera was more than 1 case per 1000 population and where approximately 35 cases per year would be expected in the placebo-control

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group, initiation of the field trial with high enrolment was followed by a precipitous fall in the expected number of cases of cholera. This could be attributed to a secular year-to-year change. However, another possible explanation may be that the field trial design (random allocation at the level of individual subject) and its performance in crowded slums with a very high population density led to a high level of indirect protection, as described by Ali et al. (56) in reanalysis of the Matlab field trial of WC-CTB vaccine. This interpretation would suggest that the combination of direct and indirect protection led to the drastic fall in cholera incidence. Thus, CVD 103-HgR may in fact have worked at the public health level even though the standard method for estimation of vaccine efficacy suggested only a modest level of efficacy with the limited number of cases that occurred in the first year of the field trial. A post-licensure efficacy evaluation of CVD 103-HgR that was tested by WHO during mass use of the vaccine in Micronesia during a cholera outbreak provided more optimistic results (77). In the course of the cholera control effort, 47% of the population received a single dose of the vaccine during a mass vaccination campaign on the island of Pohnpei. The incidence of cholera was five times greater in non-vaccinees for an estimated vaccine efficacy of 79% (95% CI of 72–85%) used under epidemic control conditions. One possible explanation for the reduced efficacy of CVD 103-HgR in subjects in the developing world is their lower postvaccination vibriocidal titers compared with titers in North American volunteers. Diminished immunogenicity of live oral vaccines given to subjects living in developing countries compared with subjects in industrialized countries has also been observed with both oral polio and rotavirus vaccines. To achieve high seroconversion rates of vibriocidal antibody in Indonesian children, it was necessary to give a dose of CVD 103-HgR 10-fold higher (5
109 CFU) than the dose (5
108 CFU) that is consistently immunogenic in North Americans and Europeans (78,79). Potential explanations for the diminished immunogenicity include small bowel overgrowth (80) and heavy infection with intestinal helminths (81). As noted in the preceding text, CVD 103-HgR is reduced in colonization ability, because of, at least in part, reduced motility and flagella expression (65), which could contribute to diminished immunogenicity. CVD 103-HgR was licensed and sold in numerous countries under the trade names Orochol1 and Mutacol1 (Berna Biotech Ltd., Switzerland) and was widely used for prevention of cholera in travelers. However, the manufacturer, Berna Biotech Ltd. (now a Crucell company) halted production in 2004.

nonmotile mutant of Peru-3 was isolated and designated Peru-15 (83); the nonmotile mutant was better tolerated in volunteers than the Peru-3 parent. A randomized, placebocontrolled clinical trial enrolled 59 North American volunteers who received 2
108 CFU of Peru-15 or placebo (84). Recipients of the vaccine and placebo had similar rates of symptoms, except for headaches, which were more frequently reported by vaccinees on days 0 and 3 (p ¼ 0.002 and 0.05, respectively), and abdominal cramps, which did not reach statistical significance. Ninety-seven percent of vaccinees mounted significant vibriocidal antibody responses and 28% mounted significant antitoxin responses. After challenge with wild-type El Tor strain N16961, 5 (42%) of the 12 placebo recipients and none of the 24 vaccinees had moderate or severe diarrhea (>3 kg diarrheal stool) (protective efficacy 100%). When mild diarrhea was included, 7 (58%) of 12 placebo recipients and 1 (4%) of 24 vaccinees had any diarrhea (protective efficacy 93%). A further derivative of this strain has been constructed to express higher levels of the CTB subunit by cloning the ctxB gene on a multicopy plasmid under the transcriptional control of a strong constitutive promoter (85). The resulting strain, Peru-15pCTB, induced antitoxin titers approximately 30-fold higher than Peru-15 when tested in an oral inoculation rabbit model. Peru-15 is currently undergoing further clinical testing in cholera-endemic countries. Initial phase I and phase II trials in Bangladesh using a single dose of 2
108 CFU showed vibriocidal antibody responses in 75% of 40 vaccinees and serum anti-CTB responses in less than 20% of subjects (86). This vaccine has also been shown to be safe and immunogenic in Bangladeshi toddlers and infants of nine months to five years (87).

V. cholerae PERU-15

Other V. cholerae O1 and O139 Vaccine Candidates

Peru 15 is an El Tor Inaba strain created from a wild-type V. cholerae O1 strain (C6709) isolated in Peru in 1991. It was constructed by deleting the ctx, ace, zot, and rtxA genes as well as the attRS attachment site for the CTX phage. In addition, the ctxB gene was cloned under the control of the htpG heatshock promoter and inserted into the chromosomal recA gene, thereby inactivating this gene involved in homologous recombination. The attenuated strain resulting from these initial genetic manipulations was called Peru-3. When tested in volunteers at doses of 4
106 and 1
108 CFU, Peru-3 stimulated significant vibriocidal antibody responses in five of six vaccinees and mild diarrhea in two of six (82). A spontaneous

V. cholerae 638 V. cholerae strain 638 is an attenuated O1 El Tor Ogawa strain that lacks both ctxA and ctxB genes. Starting with wild-type C7258, isolated in Peru in 1991, the CTXF prophage containing ctx, ace, and zot genes was deleted to yield strain 81. The hap gene encoding a hemagglutinin/protease (HA/P) was inactivated by insertion of a gene encoding an endoglucanase (celA), which provides a phenotypic marker for the strain (88). The resulting strain, 638, was tested at a dose of 109 CFU in a phase II trial in Cuba (89). Significant vibriocidal responses were seen in 96% of vaccinees along with mild diarrhea in 4 of 24 subjects. One month after immunization, 12 vaccinees and 9 placebo recipients were challenged with wild-type El Tor Ogawa strain 3008. None of the vaccinees and seven of nine controls experienced diarrhea after challenge. Further evaluation of this strain is continuing.

V. cholerae IEM101 is an O1 El Tor Ogawa strain that was isolated from a river water sample in China. It naturally lacks ctxAB, ace, and zot genes but contains tcp genes encoding the TCP pilus (90). In volunteer trials in China, this strain colonized well, no diarrhea, fever, or other side effects occurred, and serum vibriocidal antibodies were engendered. This strain has subsequently been modified to express a genetically detoxified derivative of CT (91), as well as heterologous antigens such as fragment C of tetanus toxin and tracheal colonization factor from Bordetella pertussis (92). Two further derivatives of this strain, IEM108 and IEM109, have been constructed, which

Chapter 49: Oral Cholera Vaccines

contain an rstR gene that confers resistance to the CTXF phage (93,94). No human data on the ability of these strains to protect against disease have yet been reported for these strains. Two recombinant live attenuated O139 vaccine candidate strains have been constructed and evaluated in volunteer trials. These strains, CVD 112 (95) and Bengal-15 (96) were well tolerated and conferred 83% to 84% protection against challenge with wild-type O139 strains. The possibility of combining live attenuated O1 and O139 strains into a bivalent vaccine has not been assessed, and due to the virtual disappearance of the O139 serogroup (at least as of this writing), these strains have not been further evaluated.

CONCLUSIONS Epidemiologic indications for vaccination against cholera include (i) protection of populations at risk in high-endemic areas; (ii) protection of vulnerable populations in high-risk situation (e.g., refugees); (iii) as an adjunct control measure in large cholera epidemics; and (iv) prevention of cholera in travelers. At least 120,000 persons, and probably significantly more, die each year from cholera, reflecting a failure of health systems infrastructure and difficulties in implementation of control measures. WHO has recently concluded (3) that although the establishment of adequate personal hygiene, food safety, and sanitation remain the mainstay of cholera control, the short term drastic improvements in these fields are difficult to achieve in many cholera-endemic areas, and there is therefore an urgent need for use of efficient vaccines as an additional public health tool for cholera prevention. Compared with the previous parenteral vaccines, the internationally licensed oral cholera vaccines represent significant improvements in terms of protective efficacy, duration of protection, safety, and ease of administration (3). In accordance with this, and on the basis of an extensive inquiry among a large number of global vaccine experts undertaken by WHO, cholera vaccine is now also among the top five vaccines identified by WHO and the Global Alliance for Vaccines and Immunization (GAVI) Alliance as lead candidates for GAVI-supported implementation in countries with high endemicity and/or at high risk of epidemic outbreaks. This development is based on an improved understanding of the epidemiological situation with regard to cholera, the documented safety and efficacy of available oral cholera vaccines, the promising developments in local manufacturing of oral cholera vaccines, and the recent evidence of strong herd protection that could markedly increase the overall impact of oral cholera vaccine as a tool in the control of cholera in-endemic settings. In addition to routine use in high-endemicity and large outbreak settings, the other indication for using cholera vaccines is in emergency situations involving high-risk populations, such as refugees in primitive camps and urban slum residents. Both the oral killed and live, attenuated vaccines have been shown to be logistically compatible with use in mass vaccination campaigns and have given promising results with regard to their effectiveness. WHO emphasizes that the use of cholera vaccines should ideally be preemptive before an outbreak has started. However, in reality during large epidemics in certain ecologies, there is a great demand for vaccine, particularly if a single dose would suffice. If vaccine is used in cholera control, it must always be combined with the other prevention control measures recommended by WHO.

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A last indication for cholera vaccination is for prophylactic use in civilian and military personnel traveling to choleraendemic areas (3). Since these populations can afford to pay much higher prices for cholera vaccines than those in developing countries, this indication could subsidize use of cholera vaccines in the other populations. As with other diseases that primarily affect people in the most impoverished countries, widespread use of cholera vaccines will greatly depend upon finding the financial resources to fund vaccination programs.

ACKNOWLEDGMENTS We acknowledge with gratitude the invaluable contributions by our many coworkers in the cited studies of our own published work. Jan Holmgren gratefully acknowledges the financial support from the Swedish Science Council, the Swedish Agency for Research Cooperation (SIDA) and the Wallenberg Foundation, and James B. Kaper acknowledges support from the NIH. We also thank Ms. Helene Eliasson for skilled secretarial assistance in the preparation of the manuscript.

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78. Suharyono, Simanjuntak C, Witham N, et al. Safety and immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR in 5-9-year-old Indonesian children. Lancet 1992; 340:689–694. 79. Simanjuntak CH, O’Hanley P, Punjabi NH, et al. Safety, immunogenicity, and transmissibility of single-dose live oral cholera vaccine strain CVD 103-HgR in 24- to 59-month-old Indonesian children. J Infect Dis 1993; 168:1169–1176. 80. Lagos R, Fasano A, Wasserman SS, et al. Effect of small bowel bacterial overgrowth on the immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR. J Infect Dis 1999; 180:1709–1712. 81. Cooper PJ, Chico ME, Losonsky G, et al. Albendazole treatment of children with ascariasis enhances the vibriocidal antibody response to the live attenuated oral cholera vaccine CVD 103HgR. J Infect Dis 2000; 182:1199–1206. 82. Taylor DN, Killeen KP, Hack DC, et al. Development of a live, oral, attenuated vaccine against El Tor cholera. J Infect Dis 1994; 170:1518–1523. 83. Kenner JR, Coster TS, Taylor DN, et al. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172:1126–1129. 84. Cohen MB, Giannella RA, Bean J, et al. Randomized, controlled human challenge study of the safety, immunogenicity, and protective efficacy of a single dose of Peru-15, a live attenuated oral cholera vaccine. Infect Immun 2002; 70:1965–1970. 85. Roland KL, Cloninger C, Kochi SK, et al. Construction and preclinical evaluation of recombinant Peru-15 expressing high levels of the cholera toxin B subunit as a vaccine against enterotoxigenic Escherichia coli. Vaccine 2007; 25:8574–8584. 86. Qadri F, Chowdhury MI, Faruque SM, et al. Randomized, controlled study of the safety and immunogenicity of Peru-15, a live attenuated oral vaccine candidate for cholera, in adult volunteers in Bangladesh. J Infect Dis 2005; 192:573–579. 87. Qadri F, Chowdhury MI, Faruque SM, et al. Peru-15, a live attenuated oral cholera vaccine, is safe and immunogenic in Bangladeshi toddlers and infants. Vaccine 2007; 25:231–238. 88. Benitez JA, Garcia L, Silva A, et al. Preliminary assessment of the safety and immunogenicity of a new CTXF-negative, hemagglutinin/protease-defective El Tor strain as a cholera vaccine candidate. Infect Immun 1999; 67:539–545. 89. Garcia L, Jidy MD, Garcia H, et al. The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers. Infect Immun 2005; 73:3018–3024. 90. Liu YQ, Qi GM, Wang SX, et al. A natural vaccine candidate strain against cholera. Biomed Environ Sci 1995; 8:350–358. 91. Fontana MR, Monaci E, Yanqing L, et al. IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin. Vaccine 2000; 19:75–85. 92. Chen I, Finn TM, Yanqing L, et al. A recombinant live attenuated strain of Vibrio cholerae induces immunity against tetanus toxin and Bordetella pertussis tracheal colonization factor. Infect Immun 1998; 66:1648–1653. 93. Liang W, Wang S, Yu F, et al. Construction and evaluation of a safe, live, oral Vibrio cholerae vaccine candidate, IEM108. Infect Immun 2003; 71:5498–5504. 94. Yan M, Liu G, Diao B, et al. A Vibrio cholerae serogroup O1 vaccine candidate against CTX ET Phi infection. Vaccine 2007; 25: 4046–4055. 95. Tacket CO, Losonsky G, Nataro JP, et al. Initial clinical studies of CVD 112 Vibrio cholerae O139 live oral vaccine: safety and efficacy against experimental challenge. J Infect Dis 1995; 172: 883–886. 96. Coster TS, Killeen KP, Waldor MK, et al. Safety, immunogenicity and efficacy of live attentuated Vibrio cholerae O139 vaccine prototype. Lancet 1995; 345:949–952.

50 Novel Vaccines Against Tuberculosis Marcus A. Horwitz Department of Medicine, University of California Los Angeles, Los Angeles, California, U.S.A.

Peter Andersen Department of Infectious Diseases Immunology, Staten Serum Institut, Copenhagen, Denmark

Stefan H. E. Kaufmann Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

THE TB PROBLEM AND NEED FOR A VACCINE Tuberculosis (TB) is responsible for two million deaths and nine million new cases of pulmonary TB each year. Although these numbers place TB among the most important global health problems, active disease only represents the tip of the iceberg as it has been estimated that one-third of the world’s population is latently infected with Mycobacterium tuberculosis (MTB), the primary causative agent (1). TB is particularly prevalent in developing regions of the world such as subSaharan Africa and Southeast Asia, where it is further fuelled by the human immunodeficiency virus (HIV) epidemic, and is overwhelming the limited resources that many countries have available to identify and treat active contagious pulmonary disease. As both HIV and TB target primarily the adult working part of the population, these two diseases represent major roadblocks to healthy economic development in many developing countries. Drugs against MTB have been available for more than 50 years, but treatment demands a complicated and exceedingly long-lasting treatment regimen. The World Health Organization (WHO) has initiated the directly observed therapy (DOTS) campaign in many regions, but so far this program has failed to control the global TB epidemic or prevent the rising rates of multidrug resistant (MDR) strains of MTB, which in some regions, for example, parts of Russia, are responsible for a large proportion of TB cases (1). M. bovis Bacille Calmette–Gue´rin (BCG), the only vaccine currently available against TB, is named to honor Albert Calmette and Camille Gue´rin who developed this vaccine strain between 1906 and 1919 (2), attenuating it by passaging it 230 times on potato slices first with and then without ox gall. The strain was found to be both safe and effective in guinea pigs, rabbits, and nonhuman primates. We now know that during its attenuation, BCG lost a large number of gene segments clustered in numerous regions of difference (3). This ancestral BCG strain was distributed to numerous institutions all over the world; as a result of different culture and preparation conditions over several decades, current strains of BCG differ from each other (3,4). Today, approximately 4 billion people have received BCG, which makes this vaccine the most widely used vaccine worldwide. Because of its proven efficacy in preventing miliary TB in toddlers, BCG is

part of the expanded program of immunization (EPI) promoted by the WHO. However, while the vaccine is wellestablished, discussion of its benefits and drawbacks has never ceased and includes safety aspects, interference with the tuberculin skin test as a diagnostic reagent and, in particular, the fact that although it is credited with helping to end the TB epidemic in Europe, the efficacy of this vaccine generally has been very disappointing in trials conducted in the developing world (5). In various trials, estimates of its efficacy against adult pulmonary TB have ranged from 0% to 80%, and in general, the lowest efficacy has been found in the countries with the highest incidence of skin test positivity to tuberculin, presumably due to latent TB and exposure to atypical mycobacteria in the environment (5,6). Initially, BCG vaccinations were restricted to tuberculin-negative individuals, but studies coordinated by the WHO indicated that it was safe to give BCG to those who had already converted their skin test; consequently, there was approval for mass vaccination of all age groups in TB-endemic areas. Today, a consensus has developed that BCG, although safe in all immunocompetent individuals, efficiently protects only skin-test negative individuals (primarily children) (7,8). Many explanations have been suggested, but recent studies in animal models have demonstrated that a preexisting immune response against mycobacterial antigens shared in BCG prevents the necessary BCG replication and vaccine take (6). Therefore, vaccine protection against adult pulmonary TB in high endemic countries is very limited, as was most clearly demonstrated by the 15-year follow-up data from the large multicenter trial of BCG in Chingleput, India (9). This unresolved problem has highlighted the need for novel TB vaccines. With increasing investment from public funds such as the European Union (EU), National Institutes of Health (NIH), and the Gates Foundation in recent years, TB vaccine research, development, and testing has now become a very active area, conducted mostly by public research organizations and public/private partnerships. Recent reanalysis of the commercial value of a novel TB vaccine may result in a larger investment from private industry in the future and thereby a more efficient and streamlined development of novel vaccines for this global health emergency (10).

Chapter 50: Novel Vaccines Against Tuberculosis

In this chapter, we review the principal TB vaccine strategies, status of current efforts, and discuss in detail some of the leading candidate vaccines currently in or entering clinical trials.

IMMUNITY TO TB Biology and Immunology of MTB MTB is a highly robust microbe capable of surviving in one of the most hostile of mammalian cells (11,12), the mononuclear phagocyte (MP), which is capable of killing a vast array of bacterial pathogens. Phagocytosis of microbes by MP results in the formation of phagosomes, which further mature from an early to a late stage and then fuse with lysosomes. Release of reactive oxygen and nitrogen intermediates together with discharge of lysosomal enzymes into the late phagosome destroys many bacterial pathogens. To counteract this, MTB arrests phagosome maturation at an early stage and prevents phagosome acidification (13). The neutral pH of the early phagosome provides a resource-rich milieu for MTB, giving access to nutrients as well as essential ions, notably iron. As a result, MTB replicates in resting macrophages. Even highly activated macrophages fail to achieve sterile eradication of the MTB predators, although they can block their multiplication and induce a state of dormancy (14). Once the activation status of MP is lowered, however, mycobacteria may resuscitate, leading to disease reactivation (11,15). This scenario is not solely a matter between MP and MTB, as it occurs in the lung where macrophages form productive granulomas under the guidance of T lymphocytes (12,16). The productive granuloma comprises MP of different maturation stages, from freshly immigrating monocytes to giant cells arising from fusion of several infected macrophages

Figure 1 The different ways of antigen presentation in tuberculosis.

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(12,16). Resuscitation and reactivation of MTB occur once the delicate balance between MP activated by T cells and MTB is tipped in favor of the pathogen (14,15), culminating in the development of caseous cavitaries in which MTB multiplies unrestrained in the cellular detritus. Although T lymphocytes are the major mediators of protection against TB, high titers of antibodies with specificity for numerous proteinaceous and nonproteinaceous mycobacterial antigens can be measured in sera of patients (17–19). Such antibodies have several potential antimycobacterial functions. Antibodies may attack free-living MTB, although this is a rare situation because MTB mostly resides within MP. However, with dissemination of free MTB from the primary site of implantation to other tissue sites, the pathogen may be vulnerable to attack by antibodies (20). Antibodies could also synergize with phagocytes since antibody binding to the Fc-receptor can induce potent effector mechanisms, including generation of reactive oxygen and nitrogen intermediates (21,22). Finally, preexisting antibodies in the lung could clear the few bacteria that enter the host before they can hide within macrophages. A potential new vaccine approach would be to attempt to stimulate high titers of IgA and IgG antibodies capable of eliminating MTB promptly after its inhalation and prior to its engulfment by alveolar macrophages (23). Current vaccine design focuses on stimulation of a highly potent T-cell response in an attempt to contain or even eradicate MTB after it has established itself in the phagosome of macrophages (24) (Fig. 1). CD4 T cells are generally termed T helper (Th) cells because they help other cells to perform their functions in the best possible way (25) (Fig. 2). The Th cells further segregate in different subsets, all characterized by a distinct phenotype and a unique pattern of secreted cytokines, although some overlap in cytokine secretion occurs (Fig. 2).

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Figure 2 Stimulation of different CD4 T cells and their main biological functions. Source: From Ref. 52.

However, during their maturation, Th cells may change their cytokine secretion pattern. Until recently, only two Th cell populations were known, named Th1 and Th2 cells (26). Th2 cells help in antibody production and defense against helminths. Once activated, they produce the cytokines interleukin-4 (IL-4), IL-5, and IL-13, which primarily act on basophils, eosinophils, and B lymphocytes. Th2-cell stimulation is promoted by IL-4. IL-12, produced by infected MP and dendritic cells (DC), promotes development of Th1 cells, which produce interferon gamma (IFN-g), tumor necrosis factor (TNF), IL-2, and granulocyte-monocyte colony-stimulating factor (GMCSF). IFN-g and TNF activate macrophages, IL-2 activates CD8 T cells, while GM-CSF’s role in defense against TB remains unclear. IFN-g also promotes Th1 cell development but blocks generation of Th2 cells. In contrast, IL-4 favors Th2 cell development but blocks that of Th1 cells. Tumor growth factor (TGF)-b has been known for a long time but only more recently has been demonstrated to play a role in Th cell polarization. TGF-b alone favors the development of suppressive T cells termed T regulatory (Treg) cells, which produce IL-10 and TGFb, both inhibitory for many T cells (27,28). In the presence of IL6, however, TGF-b favors generation of Th17 cells, which are then sustained by IL-23 (29–31). In contrast, IL-27 blocks differentiation of Th17 cells, and at the same time favors development of Th1 cells. Th17 cells produce IL-17 and IL-6 and have been associated with pathologic inflammation, but they also seem to play a role in defense against extracellular bacteria (29–31). With respect to TB, Th1 cells are critical to protective immunity and also contribute to pathogenesis. The role of the

Th1-associated cytokines IL-12, IFN-g, and TNF has been wellestablished, with most convincing data using knockout (KO) mice deficient in IFN-g or TNF signaling (32–35). Reactivation of TB in rheumatoid arthritis patients treated with antibodies interfering with TNF-a signaling demonstrated the important role of this cytokine in controlling latent MTB infection in humans (36). TGF-b also participates in the formation of the fibrotic wall around granulomatous lesions, and hence participates in immunity to TB (37,38). Th2 cells are probably of little value, and due to the production of IL-4, which impairs Th1 responses, may even be harmful (39). Indeed, increased proportions of Th2 cells have been described during active TB. The role of Th17 cells in TB remains to be established (40,41). Recent experiments in the mouse model have identified g/d T cells of main producers of IL-17 in TB (42). It is interesting in this context that earlier studies using IL-6 KO mice found a role for IL-6 in protection against a high load of MTB (43). Finally, in an adoptive transfer model, depletion of regulatory T cells greatly enhanced protection afforded by CD4 T cells (44). Treg cells have also been identified in TB patients (45–48). CD8 T cells also produce cytokines, notably IFN-g and TNF, and they attack infected macrophages. Concomitant with lysis of infected host cells, perforins and granulysins produced by these cytolytic T lymphocytes (CTLs) can also attack MTB directly (49,50). CD8 T cells protect through killing of MTB. Whether it is this mechanism or the secretion of IFN-g and TNF or both, CD8 T cells are known to contribute to protective immunity, particularly at later stages of infection (15). In TB, the roles of effector T cells, memory T cells, and terminally differentiated effector T cells, remain incompletely

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Figure 3 Development of immunologic memory during tuberculosis.

understood (Fig. 3). Development and maintenance of memory is promoted by the cytokines IL-2, IL-7, and IL-15, which are all members of the same family, and central memory T cells are stimulated by these cytokines to differentiate into terminally differentiated effector T cells (51). Because MTB persists in the host, it could be argued that continuous stimulation provided by mycobacterial antigens will sustain rapid activation of rapidly dividing effector T cells (52). Yet, in the peripheral blood of latently infected individuals, both memory T cells and terminally differentiated effector T cells have been identified (53). Some evidence suggests that early effector T cells preferentially produce IFN-g only whereas terminally differentiated effector T cells are capable of producing multiple cytokines, notably IFN-g, TNF, and GM-CSF and therefore, may possess greater protective efficacy (52). Future studies will be required to define more precisely these T-cell sets and the cytokines they produce, and to correlate them with protection and pathogenesis in TB. In the context of TB, directing T cells into the lung and then attracting them to the productive granuloma is of particular importance. In other systems, both effector memory T cells and central memory T cells have been identified in the airways, thus softening the segregation between these two phenotypes (54). In contrast to other tissue sites, we know only little about the chemokines and adhesion molecules that direct the trafficking of T cells to the lung (55). Yet, it appears that the chemokine CCL5 (RANTES) and the homologous receptor CCR5 on T cells are interesting candidates for lung-specific T cells (54). Similarly, CXCR3 and its ligands CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC) are also candidates for lung accumulation of T cells (54). A very recent study has provided evidence in experimental TB of mice that Th17 cells are among the first

T cells to enter the lung in TB and then trigger the chemokines CXCL9, CXCL10, and CXCL11. These chemokines attract CD4 T cells of Th1 type, which then restrict growth of MTB (41). Despite limited knowledge about the chemokines and the adhesion factors involved in T-cell migration to the lung, it will be important for any vaccine to induce T cells that can enter the lung to contact and combat MTB, either at the stage of invasion or latent infection.

TYPES OF TB VACCINES More than one type of TB vaccine is likely to be needed to reduce the global burden of TB. Four main types of vaccines have been explored in preclinical studies in animal models (Tables 1 and 2) and in clinical studies. First, a more potent ‘‘prime’’ vaccine to replace BCG in newborns and nonimmunized tuberculin-negative adults is a high priority (Table 3). Such a vaccine must not only be more potent than BCG but at least as safe. Second, a booster vaccine is needed for individuals who have already been immunized with BCG (Table 4). Such a vaccine will need to be a heterologous booster vaccine because homologous boosting with BCG appears ineffective in both preclinical and clinical studies (56–62). Heterologous primeboost strategies incorporating a replacement vaccine for BCG as the prime, such as a more potent recombinant BCG (rBCG) vaccine, are also being explored. Third, a postexposure vaccine that can boost the immunity of individuals already exposed to MTB is needed. Whether the booster vaccine for BCG (or for a BCG replacement vaccine) noted above can also serve this role or whether a different type of vaccine would be more efficacious in the postexposure setting remains to be determined. Finally, a

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Table 1 Comparisons of Various Animal Models Used for Efficacy Testing of TB Vaccines Characteristic

Mouse

Guinea pig

Cynomolgus monkey

Resemblance of the disease to human TB Genetic diversity Cost Availability of immunologic reagents Evolutionary closeness to humans Standardization of model

Low Inbred Low High Low High

High Outbred Moderate Low Low High

High Outbred High Moderate High Moderate

Table 2 Comparison of Tuberculosis in Humans and in Major Animal Models Characteristic

Human

Guinea pig

þ

þ



þ

þ þ

þ þ

 

þ

þ þ

þ þ

 

þ/ þ

Susceptibility to low doses of aerosolized M. tuberculosis High tuberculin sensitivity DTH characterized by dense mononuclear cell infiltrate Langhans giant cells in lesions Caseation necrosis

Mouse

Cynomolgus monkey

Table 3 Prime Vaccines Against Tuberculosis Significantly More Potent than BCG in Animal Models Criteria by which significantly greater efficacy than BCG demonstrated Animal model

Vaccine

Year

Mechanism

RBCG30

2000

BCG::RD1-2F9

2003

M. tuberculosis 103drrC RBCGDureChly

2004

RBCG38

2005

M. tuberculosis SO2 rBCG(mbtB)30

2006

:Expression Antigen 85B :Expression CFP10 þ ESAT-6 Attenuated M. tuberculosis Phagosome perforation :Expression 38 kDa protein Attenuated M. tuberculosis :Expression Antigen 85B; replicationlimited

2005

2007

;CFU Lung

;CFU Spleen

;Pathology

:Survival

Laboratory

þ

þ

þ

þ

M. Horwitz

67,68

þ

S. Cole

74

Guinea pig Mouse; Guinea pig

Reference

Mouse

þ

þ

J. Triccas

88

Mouse

þ

þ

S. Kaufmann

79

Y. LopezVidal C. Martin

77 83

M. Horwitz

97

þ

Mouse þ

Guinea pig þ

Guinea pig

þ

þ

Table 4 Booster Vaccines Against Tuberculosis That Significantly Enhance the Level of Protective Immunity Conferred by Parentally Administered BCG in Animal Models Criteria by which significantly greater efficacy Than BCG alone demonstrated Vaccine

Year

Antigen

r30 (Antigen 85B) MVA85A þ Fowlpox 85A Ag85BESAT-6

2005

Antigen 85B Antigen 85A

2005

2006

Antigen 85B þ ESAT-6

Animal model Guinea pig

;CFU Lung

;CFU Spleen

þ

þ

;Pathology

þ

Guinea pig

Mouse

:Survival

þ

þ

Abbreviations: BCG, Bacille Calmette–Gue´rin; CFU, colony-forming unit; MVA, modified vaccinia virus Ankara.

Laboratory

Reference

M. Horwitz

99

A. Hill

102

P. Andersen

107,108

Chapter 50: Novel Vaccines Against Tuberculosis

therapeutic vaccine has been contemplated as an adjunct to antibiotics in the therapy of TB. BCG has an excellent safety record in immunocompetent individuals and a pretty good safety record even in HIVinfected persons (63); however, BCG can occasionally disseminate in immunocompromised persons, including AIDS patients, and cause serious and even fatal disease. Ideally, new vaccines would be as safe as BCG in immunocompetent persons and even safer in immunocompromised persons.

Vaccines Replacing BCG BCG protects against childhood TB and disseminated forms of TB, such as meningitis and miliary TB (64,65). However, the efficacy of BCG against adult pulmonary TB, the most common form, has been highly variable (66). A large meta-analysis concluded that the efficacy of BCG is approximately 50% (64); however, this number obscures the fact that efficacy tended to be bimodal in epidemiologic studies, that is, the vaccine seemed to work well or not at all. In any case, there is need for a more potent and consistent vaccine to replace BCG. All the leading candidate vaccines to replace BCG are live mycobacteria. While a large number of subunit vaccines have been tested for efficacy against MTB challenge in animal studies, including protein/adjuvant vaccines, lipid vaccines, DNA vaccines, and killed mycobacteria, none of these has ever been demonstrated to be more potent than BCG in animals, especially the stringent guinea pig model. In endemic areas of the world, health care providers are loath to allow any vaccine to replace BCG in newborns that is not at least as potent as BCG. For this reason, subunit vaccines are unlikely to supplant BCG as a first vaccine in newborns. Several types of replacement live mycobacterial vaccines have been proposed, as described in the following sections. Recombinant BCG Expressing MTB Proteins These vaccines utilize BCG as a vector to deliver immunoprotective proteins of MTB. The fact that these vaccines comprise BCG enhances their acceptability as a replacement vaccine, since health care providers are reluctant to abandon BCG, except for something likely to be at least as efficacious. The acceptability of such vaccines is also enhanced by the fact that BCG has a very well-established safety profile, having been administered to approximately 4 billion persons. The first rBCG vaccine expressing MTB proteins, and the first vaccine demonstrated more potent than BCG, was rBCG30, an rBCG vaccine overexpressing the MTB 30 kDa major secretory protein, a mycolyl transferase also known as antigen 85B (67–70). The enhanced efficacy of rBCG30 was demonstrated in the demanding guinea pig model of pulmonary TB. It significantly reduced the number of lung lesions and the extent of lung pathology, markedly and significantly reduced the burden of MTB in the lung and spleen, and significantly prolonged survival compared with the parental BCG Tice strain (67,68). The development of this vaccine followed from previous studies, showing that immunization with extracellular or secreted proteins of intracellular pathogens induces potent protective immunity against challenge with the relevant pathogen, first demonstrated for Legionella pneumophila (71,72) and subsequently for MTB (73). Subsequently, a rBCG vaccine expressing two other MTB extracellular proteins, CFP10 and ESAT-6, which are in the RD1 region of BCG that was deleted from its genome during

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attenuation from M. bovis, was shown in one experiment to be more potent than BCG in the guinea pig model, reducing the burden of MTB in the spleen but not in the lung (74). This vaccine, however, was more virulent than BCG, and clinical development of the vaccine has not proceeded. A potentially safer alternative recombinant vaccine expressing the same extracellular proteins in an M. microti host has also been tested in preclinical studies; however, the potency of this vaccine in the guinea pig model was not significantly different from BCG (75). A similar rBCG vaccine expressing only the ESAT-6 protein was tested in mice, but it did not provide greater protection than BCG, either by itself or as part of a fusion protein linked to the hsp60 protein (76). rBCG vaccines expressing other MTB extracellular proteins have also been reported. rBCG vaccine overexpressing a secreted MTB 38 kDa glycoprotein was tested in mice and found to prolong survival in one experiment (77). An rBCG expressing MTB 72f, a hybrid of two proteins, was tested in cynomolgus monkeys and appeared to induce marginally better protection than BCG, although differences between the two vaccines were not significant in this small study (78). Recombinant BCG Escaping the Phagosome A second strategy employs an rBCG vaccine that secretes listeriolysin, lyses the phagosomal membrane, and allows antigen translocation into the cytoplasm of the host cell (79). The rationale underlying this vaccine, as described below, is to enhance antigen presentation of BCG antigens and induce a more rigorous T-cell response against MTB. This vaccine induced efficacy superior to BCG in mice challenged with virulent MTB of the Beijing/W genotype family. MTB Auxotrophs Another strategy utilizes attenuated mutants of MTB as vaccines (80–88). The rationale for these vaccines is that they more closely resemble MTB than BCG. Differences between M. bovis, from which BCG is derived, and MTB are slight, as these strains are 99.9% similar at the DNA level (89). However, approximately 98 genes present in MTB are absent in M. bovis; moreover, during attenuation, BCG lost approximately 38 genes, some of which may contribute to immunoprotection against MTB (3). A number of attenuated strains of MTB have been tested (80–88). Most are no more potent than BCG, and some are less potent; however, one was found more potent than BCG in the mouse model (88) and another more potent in the demanding guinea pig model (83). Attenuated MTB present substantial safety concerns because of the possibility of reversion to virulence. Their clinical investigation will likely require multiple independent attenuating gene deletions to insure their safety (90); such additional attenuations are likely to reduce their immunogenicity. Given the safety concerns surrounding these vaccines and the failure of most to demonstrate superior efficacy to BCG, the future of these vaccines is problematic. Recombinant BCG Secreting Cytokines rBCG secreting various cytokines including IL-2, IL-18, GMCSF, and IFN-g have been shown to have enhanced immunogenicity in mice (91–95). However, such vaccines have not been demonstrated to induce enhanced protective immunity. BCG Auxotrophs and Recombinant BCG Auxotrophs The increased susceptibility of AIDS patients to disseminated infection with BCG has heightened interest in an even safer

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vaccine than BCG. This has prompted the investigation of auxotrophs of BCG and rBCG as these have been found to be safer than BCG in severe combined immunodeficiency (SCID) mouse models. Both a leucine and methionine auxotroph of BCG have been reported to induce protection against MTB challenge in guinea pigs, although protection is inferior to that induced by BCG; interestingly, the leucine auxotroph does not induce a cutaneous response to tuberculin (96). An rBCG auxotroph engineered to have curtailed growth in macrophages and the immunized host and overexpressing the MTB 30 kDa major secretory protein (antigen 85B), induced protection greater than BCG in the guinea pig model (97). This rBCG has a defect in iron acquisition, but if preincubated with iron and mycobactin before immunization, it can undergo several cycles of replication in the host. This rBCG is much safer than BCG in the SCID mouse model.

Booster Vaccines About 7 to 8 million of BCG-vaccinated individuals nevertheless develop active TB each year. A booster vaccine might augment the immunity of BCG-vaccinated people and improve their capacity to ward off active TB. Few vaccines have successfully boosted the level of protection conferred by BCG vaccination in animal studies. The MTB 30 kDa major secretory protein (antigen 85B), administered once intradermally, has enhanced significantly the protection conferred by intradermally administered BCG in the guinea pig model. This is the only booster vaccine that has proven itself capable of enhancing the protection conferred by BCG in the guinea pig model (98). Modified vaccinia virus Ankara (MVA) expressing the MTB 32 kDa major secretory protein (Antigen 85A) (MVA85A) has been demonstrated to boost the level of protective immunity in mice conferred by BCG, but only when the prime is delivered intranasally (99); intranasal delivery of BCG differs from the intradermal route by which humans are routinely vaccinated with BCG. Boosting BCG with MVA85A failed to enhance the protection conferred by BCG in the guinea pig model (100,101). However, boosting BCG sequentially with MVA85A and a recombinant fowlpox virus expressing Antigen 85A enhanced survival in guinea pigs in a single small experiment (101). The MVA85A vaccine has been evaluated in humans and is discussed further below (102). Mtb72f, a hybrid of two MTB proteins, was shown to enhance survival in the guinea pig model when coadministered with BCG (103); however, it has not been reported to enhance the protective efficacy of BCG in mice or guinea pigs when administered in a prime-boost vaccination protocol. Mtb72f failed to enhance protection conferred by BCG in a rabbit model of tuberculous meningitis (104). However, in a preliminary report, boosting BCG with Mtb72f was said to enhance survival in the cynomolgous monkey model (105). An antigen 85B-ESAT-6 hybrid vaccine (Hybrid 1) has also been tested in a prime-boost regimen. It has been shown to enhance the level of protection conferred by BCG when delivered intranasally in the mouse model (106,107). The H1 did not induce greater protection than BCG in a small study in a high dose challenge exploratory guinea pig model that was tested in the EU TB Vaccine Cluster (100). The very similar vaccine based on antigen 85B-TB10.4 (H4) administered in IC31 was recently tested as a BCG booster in a large guinea pig experiment with

30 animals in each group and was found to prolong guinea pig survival after MTB aerosol challenge (Skeiky and Sadoff, personal communication). Finally, a DNA vaccine encoding MTB Antigen Rv3407 was tested in a mouse model in which BCG was administered intravenously and the DNA vaccine subsequently administered twice; boosting with this vaccine slightly enhanced the level of protection conferred by BCG (108).

Postexposure Vaccines Most people exposed to MTB contain the infection and never develop active TB. However, in about 10% of exposed individuals, active disease ensues, either soon after exposure (primary TB) or after a period of latency (reactivation TB) that may last for years or even decades. Hence, people exposed to MTB might benefit from a postexposure vaccine that would help keep the latent MTB bacteria within them in check and diminish the likelihood of reactivation TB. In essence, a postexposure vaccine is a booster vaccine for those individuals whose immunity has been primed by exposure to MTB. Whether booster vaccines akin to those discussed in the previous section also would serve as efficacious postexposure vaccines or whether specially designed vaccines are needed to combat latent MTB infection is a matter of conjecture. One strategy for a vaccine especially designed to suppress latent MTB is a vaccine comprised of proteins expressed by MTB during latency, for example, a-crystalline (HspX) (109,110). Whether postexposure vaccines comprised of such latencyexpressed proteins would be more efficacious than vaccines comprised of proteins expressed during active disease is unknown. The evaluation of vaccines for efficacy in preventing reactivation TB is cumbersome. Such studies generally utilize the Cornell model or variations thereof, in which animals are sequentially (i) infected with MTB; (ii) treated with antibiotics to reduce the infection to a low level, a state thought to mimic latency; (iii) vaccinated with the test vaccine; and (iv) immunosuppressed, typically with dexamethazone, to reactivate TB. To what extent this model recapitulates latency and reactivation in humans is unclear. A cocktail of 10 MTB antigens encoded by DNA constructs gave very modest protection in a modified Cornell model, not significantly different from the vector control (111). A second DNA vaccine encoding the M. leprae hsp60 protein initially appeared highly efficacious in a mouse model (112). However, in a subsequent report, vaccination of mice with DNA encoding the same M. leprae hsp60 protein was ineffective both in a prophylactic mode and in a Cornell model of latent TB. Additionally, when given in an immunotherapeutic mode, the vaccine induced a severe Koch-like reaction, characterized by cellular necrosis throughout the lung granulomas (113). A similar reaction was observed when a DNA vaccine encoding Antigen 85A was administered in an immunotherapeutic mode (113). In a separate report, prophylactic immunization with DNA vaccines encoding the MTB hsp60 or hsp70 heat shock proteins were not protective in mice or in guinea pigs, and guinea pigs vaccinated with the vaccines exhibited a moderate to severe necrotizing granulomatous bronchointerstitial pneumonia with bronchiolitis (114). These reports have cast doubt on both the safety and efficacy of DNA vaccines encoding heat shock proteins.

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Therapeutic Vaccines Therapeutic vaccines have a potential role as adjunctive therapy against TB. The emergence of MDR strains of MTB, including strains resistant to nearly all conventional antibiotics used to treat TB, may strengthen the rationale for such a vaccine. Preclinical trials of therapeutic vaccines typically entail challenging animals with MTB, and after a period of time, vaccinating the animals one or more times with the vaccine being tested. Therapeutic vaccines have generally not fared well in preclinical studies. As noted in the previous section, a DNA vaccine encoding the M. leprae hsp60 protein was found highly efficacious in an initial study but ineffective and potentially dangerous in subsequent studies. A DNA vaccine encoding the MTB Antigen 85A and a vaccine comprising a crude extract of MTB extracellular proteins were ineffective at reducing the burden of MTB in the lungs of mice; however, these vaccines reduce the burden of MTB in the spleens of the mice (115). In the same study, BCG had no immunotherapeutic benefit (115). In human studies, heat-killed M. vaccae have been extensively studied as an immunotherapeutic vaccine, yielding variable results (116). Recently, particularly promising results were reported in a randomized partly blinded study conducted in Argentina in which newly diagnosed HIV-negative patients were treated with placebo or heat-killed M. vaccae administered in a three-dose regimen; all patients also received chemotherapy. Patients treated with the M. vaccae regimen showed faster and more complete clinical improvement than patients administered the placebo (117).

SELECTED VACCINES IN OR APPROACHING CLINICAL DEVELOPMENT Vaccines to Replace BCG rBCG30

Rationale. rBCG30 is a recombinant BCG vaccine expressing the MTB 30 kDa major secretory protein, a mycolyl transferase known as Antigen 85B (67). Since unmodified BCG expresses a homolog of the MTB Antigen 85B that has an identical amino acid sequence, rBCG30 in essence overexpresses this protein. It expresses five to six times as much 30 kDa protein as the parental BCG Tice strain. rBCG30 stably expresses the 30 kDa protein after repeated subculture for more than one year in broth in the absence of selective pressure and after passage through guinea pigs (67). The rationale for the selection of the MTB 30 kDa protein for expression by BCG derives from the extracellular protein hypothesis for vaccines against intracellular parasites, which holds that extracellular proteins of intracellular parasites are key immunoprotective antigens because their release inside the infected host cell makes them available for proteolytic processing and subsequent presentation on the surface of host cells as major histocompatibility complex (MHC)-peptide complexes (16,20). Such MHC-peptide complexes alert the immune system to the presence of a live bacterium within the host cell and allow T-cells capable of recognizing the complexes to exert an anti-microbial effect against the host cell, either by activating the host cell such that it inhibits the multiplication of the intracellular pathogen, or by lysing the host cell, thereby denying the intracellular pathogen its preferred intracellular niche. Three types of observations support this hypothesis. First, immunization of animals with live but not killed

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L. pneumophila and MTB induces strong protective immunity (118–121). Second, guinea pigs infected with L. pneumophila and mice and guinea pigs infected with MTB develop strong T-cell responses to secreted proteins (73,122,123). Third, immunization of guinea pigs with major extracellular proteins of L. pneumophila and MTB induces potent protective immunity against aerosol challenge with these pathogens (71–73,124–127). Importantly, the major secretory protein of L. pneumophila induces potent protective immunity despite the fact that it is not a virulence determinant in the guinea pig model, indicating that it is the processing and presentation of this molecule to the immune system rather than the neutralization of a virulence determinant that results in protective immunity (125). Of the major extracellular proteins of MTB, the 30 kDa mycolyl transferase is the most abundant protein released by MTB in broth culture, making up one-quarter of the total protein released (126). Moreover, it is among the major MTB proteins of all types expressed by MTB within human macrophages (128). Hence, macrophages infected with MTB should present a rich display of MHC-peptide complexes derived from the 30 kDa protein on their surface for T-cell targeting. The MTB 30 kDa major secretory protein is highly immunogenic (126). Immunization of guinea pigs with purified protein in adjuvant induces a strong cell-mediated immune response and protective immunity (126). Peptide mapping of the protein in humans and guinea pigs has revealed abundant immunodominant epitopes (129). The rationale for selecting BCG as a vector for the delivery of the MTB 30 kDa protein was fourfold. First, like MTB, BCG is an intracellular organism and it follows a similar intracellular pathway in host cells, residing and multiplying within a phagosome; hence, antigens released by BCG should be processed and presented similarly to antigens released by MTB and result in the generation of T cells subsequently capable of recognizing and targeting MHC-peptide complexes derived from the antigen on host cells infected with MTB. Second, BCG has an excellent safety profile. Third, the BCG vector, which is highly homologous with MTB at the DNA and protein level, provides a baseline level of protection against TB. Hence, any improvement should result in a vaccine more potent than BCG. Finally, rBCG are essentially ‘‘BCGþ’’ and thus should have high acceptability in TB endemic areas. Immunogenicity and efficacy in animal models. rBCG30 has been extensively evaluated in the guinea pig model of pulmonary TB (67–69). Guinea pigs were sham-immunized or immunized with BCG or rBCG30, challenged 10 weeks later with highly virulent (Erdman strain) MTB by aerosol, and euthanized 10 weeks after challenge for enumeration of pathology and organ burden. Compared with guinea pigs vaccinated with BCG, guinea pigs vaccinated with rBCG30 had significantly fewer lung lesions, significantly less lung pathology, and significantly fewer MTB in the lung and spleen. On average, rBCG30–immunized guinea pigs had 0.8  0.1 log fewer colonyforming units (CFU) in the lung and 1.1  0.1 log fewer CFU in the spleen than BCG-immunized animals in 15 consecutive experiments (n ¼ 280 animals total for BCG Tice and n ¼ 281 animals total for rBCG30 Tice), differences that were highly significant in each of the fifteen experiments. rBCG30 is effective in guinea pigs over a broad dose range (101–106 CFU) (70). rBCG30 was evaluated for capacity to enhance the survival of guinea pigs after challenge; rBCG30-immunized animals survived significantly longer than BCG-immunized animals (68).

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Paralleling the increased protective efficacy, rBCG30-immunized animals had significantly increased cutaneous delayed-type hypersensitivity responses and antibody responses to the 30 kDa protein (Antigen 85B) (67,69). Finally, rBCG30 protects guinea pigs against challenge with M. bovis (130) and mice against challenge with M. leprae (131). In extensive animal safety tests, rBCG30 was cleared in guinea pig organs and regional lymph nodes at the same rate as BCG (68). In mice and guinea pigs, rBCG30 exhibited no toxicity even at very high doses (68). Human studies. rBCG30 is the first live recombinant vaccine against TB to be tested in humans; it was evaluated in a randomized, controlled, double-blind phase I trial (132,133). rBCG30 administered intradermally was well tolerated and there was no significant difference in reactogenicity observed between rBCG30 and BCG Tice, the parental control strain. Volunteers were followed for nine months with a wide range of immunogenicity studies. While both rBCG30 and BCG induced significantly increased BCG-specific responses, for example, lymphoproliferative responses, CD4þ IFN-g-producing T cells, and CD8þ IFN-g-producing T cells, only rBCG30 induced significantly increased Antigen 85B-specific responses. Hence, over the course of the study, recipients of rBCG30, but not BCG, showed significant increases in Antigen 85B-specific whole blood lymphoproliferative responses; IFN-g-producing CD4þ T cells; IFN-g-producing CD8 þ T cells; IFN-g-ELISPOT responses; central memory CD4þ T cells; central memory CD8þ T cells; antibody responses by ELISA; and T cells capable of inhibiting mycobacterial intracellular growth in human macrophages. Hence, in each of the eight studies of Antigen 85Bspecific immunologic responses in human volunteers, rBCG30, but not BCG, induced statistically significant responses.

More potent and safer versions of rBCG30. rBCG(mbtB)30 BCG can rarely disseminate in an immunocompromised host including AIDS patients. Therefore, a version of the rBCG30 vaccine capable of undergoing only a limited number of replications in vivo was engineered for use in immunocompromised persons and HIV-positive individuals (97). This vaccine, rBCG(mbtB)30, was rendered defective in siderophore biosynthesis and consequently dependent upon exogenous mycobactin/exochelin for iron acquisition. In broth culture, rBCG(mbtB)30 can multiply unrestricted in the presence of mycobactin; however, in macrophages and in vivo, where mycobactin is absent, it can undergo only a few cell divisions utilizing iron stored previously during growth in mycobactin-supplemented broth culture. Because of its limited ability to multiply in vivo, rBCG(mbtB)30, in contrast to BCG, is highly attenuated in the SCID mouse model. Yet, rBCG(mbtB)30 retains its potency. While not as potent as rBCG30, it has demonstrated potency superior to BCG in the guinea pig model (97). Hence, rBCG(mbtB)30 appears to be a promising vaccine for use in HIV-positive infants and adults in the early stages of HIV infection before the immune system has deteriorated substantially, ideally in concert with anti-retroviral therapy. BCG Expressing Listeriolysin Rationale. The rationale for constructing an rBCG strain expressing a cytolysin was based on the following observations. Although BCG is capable of protecting toddlers against miliary TB, it fails to confer long-term protection, and in particular to prevent reactivation of or reinfection with MTB in adults (24). It was assumed that prevention of miliary TB in toddlers is due to containment of the pathogen, which is a function of

macrophages activated by CD4 T cells. In contrast, long-term protection against reactivation and probably also protection against reinfection in adults requires the whole T-cell armamentarium comprising, in addition to CD4 T cells, CD8 T cells and probably unconventional T cells. Hence, ways to improve immunogenicity by broadening the spectrum of mycobacteriaspecific T cells was considered a valid option for constructing a better BCG vaccine. Listeria monocytogenes, an intracellular pathogen, which egresses into the cytosol (25), is a potent stimulator of CD8 T cells. Listeriolysin (Hly), which perforates the phagosomal membrane, is the main factor promoting translocation of L. monocytogenes into the cytosol (134). Hly is a member of the oxygen-labile sulfhydryl activated cytolysins found in various gram-positive bacteria including streptolysin O produced by group A streptococci and perfringolysin O produced by Clostridium perfringens (135). These cytolysins form pores of 10- to 20-nm diameter, which are sufficient for leakage of larger molecules. The latter two cytolysins bind to the outer membrane of eukaryotic cells and lyse them. Hence, their major aim is to destroy host cells. In contrast, Hly is not active in the extracellular milieu but only in the acidic (pH 5.5) milieu of the maturing phagosome. This strict pH requirement restricts the activity of Hly to the maturing phagosome and hence adds to its safety. A further addition to the safety of Hly is the presence of a PEST-like sequence, which induces degradation of Hly soon after its appearance in the cytosolic compartment (136). Hence, Hly is active for only a short period of time. The r-BCG-expressing Hly was attenuated in that it survived in macrophages for a shorter time period than the parental BCG strain (137). Gold labeling studies indicated the presence of Hly in vacuoles, which either contained BCG or not. However, the r-BCG Hly was not found in the cytoplasmic compartment. Yet, in vitro assays revealed increased MHC I presentation of the surrogate antigen ovalbumin to CD8 T cells. Moreover, protection against challenge with MTB was greater for r-BCG Hly than for BCG, but only at later time points (>180 days). Immunogenicity and efficacy in animal models. The r-BCG DureC:Hly induced better protection in mice against MTB challenge than the parental BCG over the whole period of the experiment (*240 days) (79). At late time points (>150 days), protection induced by parental BCG against the laboratory strain MTB H37 Rv was documented as a 10-fold reduction of the bacterial load in the lung. In contrast, r-BCG DureC:Hly induced >2 log difference, that is, a 100-fold reduction of bacterial load from ca. 1 million bacteria to less than 10,000 bacteria in the lung. Members of the MTB Beijing family are currently spreading all over the globe (138). Most of them are multidrug resistant as a result of poor compliance to drug treatment. Moreover, it has been claimed that the Beijing strains have become more resistant against host immunity due to the incomplete protection afforded by BCG vaccination. In fact, in experimental mice, parental BCG induced only meager, if any, protection against MTB Beijing (79). In marked contrast, the rBCG DureC:Hly construct was still capable of causing profound protection (*2 log difference). Despite increased protection, the r-BCG DureC:Hly was safer in a model of immunocompromised mice (79). SCID mice survived for less than 30 days when infected with a high dose of parental BCG, whereas they survived for more than 80 days when infected with a high dose of r-BCG DureC:Hly. Human studies. The r-BCG DureC:Hly strain has been evaluated by German regulatory agencies and graded as a

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genetically modified organism of P1 level, making it possible to proceed toward human clinical trials. The vaccine strain has been licensed to Vakzine Projekt Management GmbH (VPM), which has initiated large-scale production according to good manufacturing practices (GMP). A clinical phase I trial of the vaccine commenced in 2008 and is expected to be completed at the end of 2009. The r-BCG DureC:Hly construct is primarily considered as a substitute for the current BCG vaccine and hence its main target population will be newborns. In addition, the vaccine strain can be considered for a heterologous prime/ boost vaccination regimen with r-BCG DureC:Hly as prime and a novel subunit vaccine as boost.

Booster Vaccines Subunit Vaccines Based on Fusion Molecules The fusion of individual vaccine antigens into polyproteins has been demonstrated to increase the immunogenicity of the individual antigens and has obvious advantages from a manufacturing point of view. The approach of combining a fusion protein and appropriate adjuvant has been used in the design of some of the leading subunit vaccines and represents a feasible strategy for a future TB vaccine that can be produced at industrial scale.

The Ag85B-ESAT6 fusion molecule. Rationale The Ag85B-ESAT6 fusion molecule (H1) is made up of two of the best characterized and extensively evaluated protective antigens from MTB. Both Ag85B and ESAT6 are secreted antigens initially identified in the early culture filtrate of multiplying bacteria (139). The focus on antigens from culture filtrates was initially based on the classical observation that live mycobacteria protected against TB but killed ones did not and the hypothesis that this was due to the loss of important antigens only produced by the dividing organism (73,124). The interest in this group of antigens was accelerated by the observation that vaccines based on complex mixtures of culture filtrate antigens administered with strong TH1 promoting adjuvants protected efficiently against TB challenge in both the mouse and the guinea pig model (73,140). Using molecular mass fractionation, it soon became clear that the majority of immune reactivity was found in the low molecular mass region below 10 kDa and in the region from 25 to 35 kDa (139). In the 25 to 35 kDa region, the previously characterized Ag85 family was identified, whereas dissection of the low molecular mass region resulted in the identification of ESAT-6. Due to its presence in culture filtrates, ESAT-6 was classified as a secreted protein but no conventional signal sequence was found (141) and only recently has a specialized secretion system that is responsible for secretion of proteins from the ESAT family been discovered (142). The Ag85 complex (A–C) consists of a family of closely related 30 to 32 kDa mycolyl transferases that are involved in cell wall biosynthesis and are among the most abundant culture filtrate components. Both ESAT6 and Ag85B are antigens that are strongly recognized in animal models of TB infection as well as in TB infected individuals (139,143,144).

Immunogenicity and efficacy in animal models Ag85A/B have been evaluated in the mouse and guinea pig models and have an impressive track record of studies confirming their vaccine potential using a variety of delivery vehicles including DNA, viruses, and adjuvants (99,126,145,146). Similar promising results have been obtained using ESAT-6. Delivered in cationic liposomes based on DDA/ MPL, it protects well in the mouse aerosol challenge model

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(147), and also as a DNA vaccine, it has shown considerable promise (148). The vaccine potential of the H1 fusion molecule was first evaluated by Olsen et al. in 2001, and it was demonstrated that when H1 was administered in cationic liposomes, it was highly immunogenic and protected mice (149) and guinea pigs (150) against aerosol challenge with TB at levels comparable or even superior to BCG. When administered as a DNA vaccine, it also provides high levels of protection (similar to BCG) in the mouse model measured in terms of both reduction in bacterial replication in animal organs and survival time (151). The immunogenicity and protection provided by the fusion molecule is clearly superior to that induced by the individual antigens in a number of different delivery systems (149,151,152) and importantly, the fusion molecule promotes an efficient long-term memory response and protection against aerosol challenge, which is sustained for up to 18 months post vaccination (149,151). H1 can be administered via the mucosal route either orally or intranasally with the mucosal adjuvants LTK63 (a modified, heat-labile enterotoxin from Escherichia coli) (106)] or CTA1-DD (cholera toxin A1-subunit fused to a protein A fragment) (107). Recently, the H1 vaccine underwent a successful evaluation in mice and guinea pigs combined with the IC31 adjuvant (Intercell AG), which is a mixture of oligodeoxynucleotides and polycationic amino acids that promotes a strong Th1 response (153). H1 has now undergone two independent evaluations in macaques with positive results measured as strong immunogenicity and efficient protection against both bacterial multiplication and pathology (154,155). Of particular importance for its future use in human populations, a number of studies have demonstrated the potential of H1 for boosting BCG. As ESAT6 is not present in BCG, in addition to boosting the Ag85B responses primed by BCG, this vaccine will expand BCG’s antigen repertoire with the ESAT6 antigen. The general experimental outline in these experiments is to wait for the activity of BCG to wane (up to 1 year post vaccination) and then boost the responses with the subunit vaccine. There are at least three recent studies demonstrating a strong booster effect of H1, and in two of these studies, the booster vaccinations were administered via the intranasal route, significantly enhancing protection conferred by BCG (106,107). In the third study, the fusion molecule delivered as a DNA vaccine was compared with a cocktail of different DNA vaccines based on individual antigens, and a strong boosting of immunity to levels observed immediately after BCG vaccination was observed only with the fusion vaccine (151). This supports the vaccine potential of genetically engineered fusion molecule antigens compared with cocktails of individual antigens. Human studies H1 is currently undergoing clinical evaluation administered both parentally and via the mucosal route. The first clinical trial in Leiden, Holland (Dissel and Ottenhoff, Leiden University Medical Center) evaluated the vaccine in a conventional parenteral vaccination strategy, using the IC31 adjuvant. This trial was conducted in Purified Protein Derivative (PPD)-negative individuals and the vaccine was shown to be both safe and strongly immunogenic. The H1/ IC31 vaccine is currently being evaluated in PPD-positive BCG vaccinated individuals at the same clinical site. Another trial has recently started in the United Kingdom (David Lewis, St. George Hospital) to test the H1 antigen by the nasal route with LTK63 adjuvant (Novartis Vaccine and Diagnostics Srl, Siena, Italy). Safety and immunogenicity trials will continue in Ethiopia in late 2007 with the aim of further analyzing safety and immunogenicity in populations at high risk of TB infection.

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The Ag85B-TB10.4 fusion molecule. Rationale In addition to being a valuable vaccine component, ESAT6 (the component of H1 that is localized in a region deleted during the original attenuation of BCG and therefore absent from all vaccine strains) is a key component in a new generation of diagnostic tests for MTB infection (156). An H1-based vaccine could, therefore, potentially compromise the diagnostic utility of these novel tests in distinguishing between infection and vaccination. Therefore, a new fusion construct, called H4, has been engineered, which consists of Ag85B and TB10.4 (157). This vaccine was developed not only to avoid interference with diagnostic tools employing ESAT 6 but also as an optimized booster vaccine for BCG. TB 10.4 belongs to the ESAT-6 gene family (158), but in contrast to ESAT6, this antigen is present in all strains of BCG and constitutes one of the antigens responded to most strongly after BCG vaccination (159). Immunogenicity and efficacy in animal models In animal models, TB10.4 is a dominant T-cell target for both effector memory T cells and long-lived central memory T cells after both BCG vaccination and TB infection (160), and the frequency of CD4 T cells specific for this antigen correlates with protection against MTB infection (161). H4 was recently evaluated in the aerosol mouse model and administered in cationic liposomes. It induced protection at BCG level, and as demonstrated for H1, H4 was clearly superior to the individual Ag components (157). In the guinea pig model, H4 administered in IC31 was recently tested as a BCG booster in a large experiment and found to prolong guinea pig survival after MTB aerosol challenge (Skeiky and Sadoff, personal communication). Having removed ESAT6 from the vaccine, this study also demonstrated a clear correlation between the magnitude of T-cell responses to ESAT6 and the progression of MTB infection; this may provide a new way of monitoring vaccine efficacy in clinical trials. Human studies H4 in the IC31 adjuvant is entering clinical phase I trials in PPD-negative and positive individuals in 2007 supported by the Aeras Global TB Vaccine Foundation.

The MTB72f vaccine. Rationale The MTB72f vaccine is a fusion molecule consisting of two proteins selected in an antigen discovery program coordinated by Reed and colleagues in which the T-cell responses of PPD-positive adult humans was used as a guide, and antigens that induced high IFN-g responses selected (162). This successful program resulted in the discovery of a large number of interesting molecules, many of which were from the PE, PPE, or ESAT families of molecules or among the serine proteases secreted by MTB. Some of these proteins have now been evaluated individually and found to give protection in animal models (163,164). A number of the hits from this program were engineered into fusions that were evaluated under the NIH TB Vaccine Contract evaluation program. One of these fusion molecules, Mtb72F, consistently gave good results in these screens. This molecule consists of a polyprotein made up of Rv1196 (MTB32) inserted into the middle of the serine protease Rv0125 (MTB39), which is thus present as two fragments (165).

Immunogenicity and efficacy in animal models MTB72F has been evaluated in combination with the AS01/ AS02 adjuvants from GSK and as a DNA vaccine and has been demonstrated to induce a strong IFN-g response and to protect both mice and guinea pigs against aerosol challenge (165). Mtb72F was also reported to induce efficient protection if coadministered with BCG and to improve the protection

compared with BCG alone in the guinea pig model (103). A similar tendency was seen in the more relevant scenario in which prior BCG vaccination was boosted with Mtb72F delivered as a DNA vaccine, and although the differences in survival or bacterial numbers did not reach statistical significance, the lesions present in these animals upon autopsy showed evidence of wound healing and airway reestablishment (103). The MTB32 part of the fusion molecule contains a CD8 CTL epitope strongly recognized in the mouse model and T cells directed to this epitope can be tracked by MHC class I tetramers after vaccination and during natural infection in the lung (166). Most recently, vaccination with Mtb72F formulated in AS02A or Mtb72F formulated in AS01B was reported to be protective against central nervous system challenge with MTB H37Rv in a rabbit model to an extent comparable to that of vaccination with BCG (104). Human studies Mtb72F in AS02A formulation has recently completed two phase I trials in healthy PPD-negative adults in the United States and Belgium and the vaccine was well tolerated and safe and could induce antigen-specific humoral and cell-mediated immune responses. Very recently, a phase I/II study has been completed in healthy volunteers who were PPD-positive either via previous vaccination with BCG or through exposure to MTB. Further trials are planned in countries where TB is endemic, with the aim of providing proof of concept of the efficacy of the vaccine candidate in populations at high risk of TB infection. MVA85A

Rationale. MVA is an attenuated strain of vaccinia virus that is unable to replicate in mammalian cells. Various MVAbased vaccine constructs have been safely administered to humans (167). MVA85A is an MVA strain expressing the 32 kDa major secretory protein of MTB, also known as Antigen 85A. This protein is a well-established immunoprotective antigen of MTB (126,168). Antigen 85A is found in all strains of BCG, allowing for a heterologous prime-boost vaccination strategy using BCG as the prime and MVA85A as the boost. Immunogenicity and efficacy in animal models. Balb/c mice primed with BCG and boosted with MVA85A developed antigen-specific CD4þ and CD8þ T cells (99). Mice primed with BCG intranasally, boosted with MVA85A intranasally or parenterally, and then challenged with MTB had markedly fewer MTB in the lung and spleen then nonboosted mice; boosting with MVA85A intranasally induced protection comparable to boosting with BCG intranasally (99). In guinea pigs primed with BCG, boosting with MVA85A did not result in enhanced protection against MTB challenge (100,101). However, boosting BCG-primed guinea pigs twice, first with MVA85A and then with a fowlpox virus expressing Antigen 85A (fowlpox85A), resulted in improved survival in guinea pigs compared with not boosting BCG-primed animals at all in one experiment (101). Human studies. In a phase 1 study in humans, MVA85A was found to be safe and well tolerated (102). MVA85A is the first subunit TB vaccine tested in humans. Immunogenicity studies focused exclusively on antigen-specific IFN-g secreting T cells. In BCG-naive volunteers, a single immunization with MVA85A induced high levels of IFN-g-secreting T-cells reacting with PPD (purified protein derivative of MTB), a complex of Antigens 85A, B, and C, or pooled peptides of Antigen 85A; the response peaked one week after vaccination. In people who had been vaccinated 0.5 to 38 years previously, immunization

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with MVA85A induced high levels of antigen-specific IFN-gsecreting T cells; 24 weeks after immunization, these levels were much higher than those in BCG-immunized controls not vaccinated with MVA85A.

CONCLUSIONS The need for better TB vaccines has never been greater as the HIV pandemic has rendered many individuals highly susceptible to TB, and the emergence of extensively drug-resistant strains of MTB (XDR-TB) has made many infections virtually untreatable. Fortunately, the last few years have seen the development and testing in animal models of recombinant vaccines more potent than the current BCG vaccine and booster vaccines capable of enhancing the level of protective immunity conferred by BCG. Several vaccines have already entered or will soon enter human clinical trials. Thus, one hundred years after Calmette and Gue´rin initiated the development of BCG, new and better TB vaccines are on the horizon.

ACKNOWLEDGMENTS Work on TB vaccines in the Horwitz laboratory is supported by National Institutes of Health grants AI068413 and AI31338. The phase 1 study of rBCG30 was sponsored by the Aeras Global TB Vaccine Foundation with the support of the Bill & Melinda Gates Foundation. Work on TB vaccines in the Kaufmann laboratory is supported by: EUPF6 TBVAC, EUFP6 MUVAPRED, Bundesministerium fu¨r Bildung und Forschung KompetenzNetzwerk PathoGenoMikPlus, DFG Priority Program Novel Vaccination Strategies and the Bill & Melinda Gates Foundation Grand Challenges (GC6, GC 12) in Global Health. Vaccine research in the Andersen Laboratory is supported by EUPF6 TBVAC, EUFP6 MUVAPRED, Grants from the Danish Research Council and the Bill & Melinda Gates Foundation Grand Challenges (GC6, GC12) in Global Health and the AERAS Global TB Vaccine Foundation.

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gammadelta and alphabeta T cell responses in primates. Eur J Immunol 2007; 37(2):549–565. Langermans JA, Doherty TM, Vervenne RA, et al. Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT6. Vaccine 2005; 23(21):2740–2750. Pai M, Riley LW, Colford JM Jr. Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004; 4(12):761–776. Dietrich J, Aagaard C, Leah R, et al. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 2005; 174(10): 6332–6339. Brodin P, Rosenkrands I, Andersen P, et al. ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol 2004; 12(11):500–508. Skjot RL, Brock I, Arend SM, et al. Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat6 gene family. Infect Immun 2002; 70(10):5446–5453. Kamath A, Woodworth JS, Behar SM. Antigen-specific CD8þ T cells and the development of central memory during Mycobacterium tuberculosis infection. J Immunol 2006; 177(9):6361–6369. Hervas-Stubbs S, Majlessi L, Simsova M, et al. High frequency of CD4þ T cells specific for the TB10.4 protein correlates with

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protection against Mycobacterium tuberculosis infection. Infect Immun 2006; 74(6):3396–3407. Alderson MR, Bement T, Day CH, et al. Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(þ) T cells. J Exp Med 2000; 191(3):551–560. Skeiky YA, Alderson MR, Ovendale PJ, et al. Protection of mice and guinea pigs against tuberculosis induced by immunization with a single Mycobacterium tuberculosis recombinant antigen, MTB41. Vaccine 2005; 23(30):3937–3945. Skeiky YA, Ovendale PJ, Jen S, et al. T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection. J Immunol 2000; 165(12):7140–7149. Skeiky YA, Alderson MR, Ovendale PJ, et al. Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein. J Immunol 2004; 172(12):7618–7628. Irwin SM, Izzo AA, Dow SW, et al. Tracking antigen-specific CD8 T lymphocytes in the lungs of mice vaccinated with the Mtb72F polyprotein. Infect Immun 2005; 73(9):5809–5816. McShane H, Pathan AA, Sander CR, et al. Boosting BCG with MVA85A: the first candidate subunit vaccine for tuberculosis in clinical trials. Tuberculosis 2005; 85:47–52. Huygen K, Content J, Denis O, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 1996; 2:893–898.

51 Influenza John Treanor Division of Infectious Diseases, Department of Medicine, University of Rochester Medical Center, Rochester, New York, U.S.A.

Wendy Keitel Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A.

Iain Stephenson Infectious Diseases Unit, Leicester Royal Infirmary, Leicester, U.K.

INTRODUCTION Influenza is widely recognized as an important target for prevention by vaccination because of the considerable yearly burden of death, hospitalization, and medically attended illness associated with influenza epidemics. Influenza epidemics are estimated to be associated with approximately 200,000 hospitalizations and 36,000 to 40,000 excess deaths in the United States (1,2), and a large burden of excess hospitalizations and other complications. The largest burden of mortality is associated with epidemics of influenza A (H3N2) (3), but influenza A (H1N1) and influenza B virus epidemics are also associated with significant disease impact. Therefore, current influenza vaccines are trivalent formulations containing components inducing immunity against all three viruses. The burden of influenza falls more severely on those at the extremes of age, with higher rates of medically attended illness and hospitalizations in children under five (4) and older adults, with hospitalizations gradually increasing at about age 50 (2). In addition, complications and deaths from influenza are of particular concern in those with cardiovascular and pulmonary conditions, or those requiring regular medical care because of chronic metabolic, renal, blood, or immune diseases (5). Influenza may also result in more severe disease and increased hospitalization rates in individuals with human immunodeficiency virus (HIV) infection (6), in those with iatrogenic immunosuppression (7), and in pregnant women, particularly in the second or third trimesters (8). However, while these conditions clearly increase the risks of severe influenza, a substantial portion of hospitalizations occur in individuals without identified risk factors. Recent observations of severe human disease caused by influenza viruses of avian origin have raised concerns regarding the imminent potential for a new pandemic of influenza. Development and implementation of more effective control measures for influenza therefore represent important research and health policy priorities. The main goal of the current strategy for use of influenza vaccine in the United States is to reduce the burden of disease by targeting vaccine to individuals at highest risk of influenzarelated medical care, hospitalizations, or death. Table 1 lists those groups for whom annual influenza vaccination is currently

recommended (9). Vaccine is recommended to reduce disease burden in high-risk individuals, and also to reduce the risk of transmission by vaccinating their family contacts and caregivers, and by vaccinating health care workers (HCWs)(10). Finally, the vaccine can be used by individuals who do not fall into a target group but who simply wish to avoid an unpleasant illness. Taken together, the current recommendations result in about two-thirds of the U.S. population falling within one or more of the target vaccination groups. Thus, it has been argued that a more simple and effective strategy could be to recommend universal annual vaccination, as has been instituted recently in the province of Ontario, Canada (11). Such a strategy could result in reduced overall burden in high-risk groups, as well, by reducing transmission in the community. There is little direct evidence supporting the use of influenza vaccine to prevent community transmission, but in one study, mass vaccination of school-aged children resulted in reduced rates of influenza in teachers and parents compared with a control community where children were not vaccinated (12). Results of a recent U.S. clinical trial suggest that vaccinating children in day care reduces the spread of influenza contacts of vaccinated toddlers (13). Similar findings have been made with cold-adapted influenza vaccine (CAIV), where indirect protection of adults was reported following vaccination of children (14). In addition, it has been observed that influenza-related mortality rates among the elderly have increased in Japan, coincident with discontinuation of that country’s policy of universal vaccination of schoolchildren (15). Rates of invasive pneumococcal disease have also fallen in adults with the initiation of universal vaccination of children against pneumococcus (16). These observations suggest that expanding the population targeted for annual influenza immunization, particularly school-aged children, could be a reasonable approach to reducing the impact of influenza in the whole community.

IMMUNE RESPONSES INVOLVED IN PROTECTION Immune responses to influenza relevant to vaccine development have recently been reviewed (17), and are schematically presented in Figure 1. Infection generates strong mucosal and

Chapter 51: Influenza Table 1 Current Target Groups for Influenza Vaccination, United States Groups at increased risk of influenza complications . Children aged 6 to 59 mo . Adults 50 yr and older . Children and adolescents (6 mo to 18 yr) receiving long-term aspirin therapy . Women who will be pregnant during the influenza season . Adults and children with chronic pulmonary, cardiovascular, renal, hepatic, hematologic, or metabolic disorders. (Note: hypertension is not a high-risk condition, but asthma and diabetes mellitus are.) . Adults and children with immunosuppression, including HIV . Adults and children with conditions compromising respiratory function or handling of respiratory secretions or increases the risks of aspiration (e.g., cognitive dysfunction, spinal cord injury, seizure disorders, or other neuromuscular disorders) . Residents of nursing homes and other chronic care facilities Groups who may transmit influenza to others who have high-risk conditions . Health care personnel . Household contacts and caregivers of children less than 5 or adults over 50, with particular emphasis on vaccination of contacts of children who are less than 6 months old . Household contacts and caregivers of persons with high-risk medical conditions Source: From Ref. 9.

Figure 1 Immune mechanisms of protection and recovery. Primary infection (shown on left) in the absence of preexisting antibody results in high levels of viral replication and severe symptoms. Replication is ultimately limited by the development of an effective cellular immune response. Reinfection with an antigenic variant of the same subtype results in more limited viral replication and attenuated symptoms, with the more rapid development of antibody and cellular responses. Source: From Ref. 17.

serum antibody responses as well as cellular responses involved in recovery from infection and protection from reinfection. In turn, influenza undergoes two evolutionary processes, antigenic drift and antigenic shift, which allow these viruses to continue to reinfect throughout an individual’s lifetime. Antigenic drift refers to the accumulation of single or multiple amino acid changes in key targets of immunity, such as the hemagglutinin (HA) and neuraminidase (NA), while antigenic shift refers to the complete replacement of these proteins with new subtypes. Antigenic shift only occurs with influenza A viruses, and is the mechanism responsible for pandemics (see below).

Serum Antibody Infection with influenza virus results in the development of antibody to the influenza virus envelope glycoproteins HA and

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NA, as well as to the structural matrix (M) and nucleoprotein (NP) proteins. Some individuals may develop antibody to the M2 protein as well (18). Serum IgM, IgA, and IgG antibody to the HA appear simultaneously within two weeks of infection (19), and the development of anti-NA antibodies parallels that of anti-HA antibodies (20). Peak antibody responses are seen at four to seven weeks after infection and decline slowly thereafter; titers can still be detected years after infection even without re-exposure. For example, during the 1976 swine immunization campaign, approximately 80% of older persons had serum hemagglutination inhibition (HAI) antibody to the H1N1 virus, despite the fact that they had not been exposed to the antigen for at least 20 years (21). Antibody to the HA, which can be measured by standard HAI tests or a variety of ELISAs, neutralizes virus infectivity (22). Anti-HA antibody protects against both disease and infection with the homologous virus (23). Although there is no exact correlation, serum HAI titers of 1:40 or greater, or serum neutralizing titers of 1:8 or greater, are associated with protection against infection in healthy adults (24), while HAI titers of 1:20 or 1:10 are associated with lesser degrees of protection (25). Antibody mediated protection is primarily strain specific, but some degree of protection is present against strains showing antigenic drift within a subtype, depending on the degree of drift (26,27). Generally, antibody which is present in low quantity or which is primarily directed against a heterologous strain of influenza may only modify the severity of illness and not prevent infection. However, establishing a specific HAI titer as a correlate of protection must take into account the variability in titers as determined in different laboratories and with different antigens. In part, this may be due to the relatively subjective nature of the visual reading of the HAI end point. In a recent study involving tests of a panel of standard human sera in 11 laboratories in eight countries, there was significant intralaboratory assay variability for the HAI test, with median geometric coefficients of variation between 138% and 261% depending on the specific virus HAI (28). Virus neutralization assays showed even greater variability. Antibody to NA can be measured by NA inhibition (NI) or ELISA. In contrast to anti-HA antibody, anti-NA antibody does not neutralize virus infectivity, but instead reduces efficient release of virus from infected cells, resulting in decreased plaque size (29) and reductions in the magnitude of virus shedding in infected animals (30,31). Observations on the relative protection of individuals with anti-N2 antibody during the A/Hong Kong/ 68 (H3N2) pandemic (20,32), as well as experimental challenge studies in humans (33), have shown that anti-NA antibody can protect against disease and results in decreased virus shedding and severity of illness, but is infection permissive (34). Antibodies to other viral proteins have also been correlated with protection in animal models. In particular, antibody to the M2 protein of influenza A virus reduces plaque size (35), and is partially protective when passively transferred to mice, similar to the effects of anti-NA antibody (36,37).

Mucosal Antibody Since the replication of influenza virus in humans is restricted to epithelial cells of the respiratory tract, it is reasonable to expect that immune responses with a mucosal site of action would be highly effective at preventing infection. Studies in mice and ferrets have emphasized the importance of local IgA

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antibody in resistance to infection, particularly in protection of the upper respiratory tract. Polymeric IgA has been shown to be specifically transported into the nasal secretions of mice, and to protect against nasal challenge. Protection can be abrogated by intranasal administration of antiserum against IgA but not IgM or IgG (38). Limited studies have demonstrated significant mucosal responses to influenza virus infection in humans, with development of both HA-specific IgA and IgG in nasal secretions. Nasal HA-specific IgG is predominantly IgG1, and correlates well with serum levels, suggesting that nasal IgG originates by passive diffusion from the systemic compartment (39). Nasal HA-specific IgA is predominantly polymeric and mostly IgA1, suggesting local synthesis. These studies in human have also suggested that the protective immunity induced by influenza virus infection can be mediated by mucosal HA-specific IgA (33,40). However, studies in IgA knockout mice (41) have shown that mucosal immunity is not required for vaccine-mediated protection, and persons with selective IgA deficiency do not appear to be at increased risk for influenza infection.

Cellular Immunity Influenza infection generates robust cellular immune responses in mice, including both CD8þ cytoxic T lymphocytes and CD4þ helper T cells. Influenza-specific, CD8þ HLA class I–restricted cytotoxic T cells lyse influenza infected cells by a variety of mechanisms, and may recognize peptide epitopes from the HA, NA, or internal proteins such as M, NP, or PB2 (42). Therefore, cytotoxic T lymphocytes (CTLs) may be subtype-specific, or in the case of those which recognize internal proteins, may be broadly cross-reactive, for example, lysing cells infected with influenza A but not influenza B virus (43–45). In mouse models, CD8þ cytotoxic T cells play an important role in limiting viral replication. However, to be effective in mediating protection in these models, they must be able to migrate to the infected respiratory epithelium quickly enough, and in large enough numbers to be able to suppress the virus before it gets out of control. In one sense, this can be seen as a race between the virus and the CTL response. CD4þ helper T cells are class II restricted, and may recognize peptides from either envelope or internal proteins. Their main function is to provide help for B cell production of antibody, and to secrete a wide array of proinflammatory cytokines. In addition, class II–restricted cells may exhibit cytotoxic activity similar to that shown by class I– restricted cells (44). Adoptive transfer experiments have shown that virusspecific T lymphocytes, including both HA-specific and crossreactive T cells, can mediate recovery from influenza virus infection in animal models. The significance of T cells directed against internal viral proteins in protection against severe disease in humans is unclear, as the internal virus proteins were shared between viruses causing the pandemics of 1957 and 1968, and the viruses in circulation immediately prior to these pandemics. However, the presence of virus-specific prechallenge class I–restricted CTLs has been shown to correlate with reductions in the duration and level of virus replication in adults with low levels of serum HA and NA antibody who were experimentally challenged with influenza A virus (46). Lymphocyte responses may play a role in ameliorating the severity of disease and speeding recovery following infection, as suggested by the finding of more severe influenza in individuals with severe defects in cell-mediated immunity (7).

In addition, during pandemics, adults who may have crossreactive T cells from previous infections appear to have some protection compared with children who have not previously had influenza, despite the fact that neither group would be expected to have antibody to the pandemic virus (47).

INFLUENZA VACCINES CURRENTLY LICENSED IN THE UNITED STATES Inactivated Influenza Vaccines Inactivated influenza virus vaccines were first licensed in the United States in 1943. Early vaccines consisted of formalininactivated whole virions grown in embryonated chicken eggs demonstrated *70% protective efficacy in healthy adults (48). Since then, although there have been several important advances in the techniques for producing vaccine, the basic vaccine strategy has remained the same. The development of the zonal gradient centrifuge allowed more efficient production and more highly purified vaccines in which reactogenic contaminants had been removed (49). Treatment of the whole virus to create split vaccines, or subunit vaccines has resulted in a vaccine with fewer adverse reactions. The efficiency of vaccine production has also been improved through the use of reassortant strains which contain the HA and NA genes from currently circulating influenza viruses, and the remaining genes from a master strain adapted to grow in high yield from hens’ eggs (50). The vaccine is currently formulated to contain at least 15 mg of each HA antigen as assessed by single radial immunodiffusion (SRID) (51), although higher-dose vaccines are being contemplated (see below). Safety Influenza vaccine is generally very well tolerated in adults. A randomized, double-blind, prospective study in over 800 healthy working adults (52) documented rates of arm soreness of 64% in vaccine recipients compared with 24% in recipients of placebo. The majority (67%) of those experiencing arm soreness after vaccination rated this symptom as mild, and only 3% rated arm soreness as severe. Rates of mild local soreness following inactivated influenza vaccine in the range of 60% to 80% have been documented in other, similar studies (53–55). Local side effects are slightly more common in women than in men (52). Among elderly persons living in the community, injection site soreness was reported more frequently in recipients of trivalent inactivated vaccine (TIV) compared with placebo recipients (20% vs. 5%, respectively) (52,56). Clinical protocols have been proposed to administer TIV to persons who are at high risk for severe or complicated influenza, who also have a history of immediate hypersensitivity to eggs, if the benefit of immunization is judged to outweigh the risk (57,58). Guillan-Barre´ syndrome (GBS), an acute inflammatory demyelinating polyneuropathy, has been associated with a variety of infectious agents, particularly Campylobacter jejuni, and occasionally develops after influenza vaccination (59). An increased risk of GBS was observed after receipt of swine influenza vaccine in 1976 (60). In subsequent years, surveillance for influenza vaccine-associated cases did not detect an obvious association, but a small risk of GBS was noted in 1992 and 1993 surveillance that would result in about one additional case of GBS per million persons vaccinated against influenza (61). The most recent studies suggest a statistically significant but very slight increased relative risk of GBS within seven weeks of influenza vaccination (62). For patients who have a history of

Chapter 51: Influenza

influenza vaccine-associated GBS who are not at high risk for influenza, it is reasonable to avoid revaccination. Immunization with TIV has not been associated with the development or exacerbation of multiple sclerosis (63). Adverse pregnancy outcomes have not been associated with influenza immunization (64,65), and immunization during pregnancy results in transplacental transfer of antibody to the infants, which may confer protection to the infant as well as the mother (66–68). It has even been suggested that maternal immunization may result in active humoral and cellular immune responses in the fetus through unknown mechanisms (69). Recent recommendations to expand the indications for routine influenza vaccination to healthy children (9) have stimulated enhanced surveillance for vaccine-related adverse events in children. The Vaccine Adverse Event Reporting System, the Vaccine Safety DataLink project, and the Clinical Immunization Safety Assessment network have been used to identify and explore issues related to vaccine safety. Available reports provide reassuring evidence of the safety of TIV among children (70–73). Vaccination of children and adults with asthma has not been associated with a significant increase in the frequency of clinical exacerbations in most studies (74,75). Immune Response Increases in HAI antibody are seen in about 90% of young, healthy recipients of inactivated influenza vaccine (76–78). Serum antibodies peak between two and four months after vaccination but wane quickly, falling to near baseline before the next influenza season (79). Mucosal anti-influenza antibodies can also be detected in 30% to 60% of immunologically primed recipients following parenteral inactivated influenza parenteral vaccine (80–83). Antibody to the HA in nasal secretions peaks between two and four weeks after immunization in primed, healthy adults and falls over the next three to six months (84). Antibody-secreting cells (ASCs) appear in blood and tonsils as early as two days after vaccination (85), and are detected in the blood of adults and older children more frequently than in young children after immunization (86). Cellular immune responses following inactivated vaccine have not been studied in detail. An increase in CTLs has been shown in healthy adults with a peak at 14 and 21 days after vaccination and return to baseline at six months (87–89). The CTL response is directed primarily toward conserved epitopes on the NP and/or M1 proteins (90). Baseline frequencies of

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influenza-specific, interferon g-producing memory CD4þ T cells are higher in children who received more previous vaccinations (91). An increase in HA-specific CD8þ T cells on day 7 after vaccination has also been detected by tetramer staining in adults receiving inactivated influenza vaccine (92). Because adults and older children have experienced many prior influenza infections and/or vaccinations with related influenza viruses, they require only a single dose of vaccine for annual immunization. However, younger children who have not previously been vaccinated are not primed and require a two-dose schedule. Studies of children who received seasonal vaccination suggest that there is an effect of strain change on priming by inactivated vaccine (Table 2). In these studies performed in young, immunologically naı¨ve children, two doses of inactivated vaccine using the same components was more effective at generating antibody than two doses of vaccine containing differing components (93). When vaccine antigens did not change in two successive years, responses among children who received the first dose of vaccine in the spring and the second dose in the fall were similar when compared with those who received both doses in the fall (94). However, in two seasons where the vaccine antigens differed, the responses to the new antigen was lower among children given the older and newer variants in the spring and fall, respectively, than those observed among children given two doses of the newer variant in the fall (95,96). Serum antibody responses are often lower among very young and elderly persons. Among infants given a purified vaccine, antibody responses among six to nine months of age were much more common than among two to six months of age (97). Reduced responses among young children may be related to a combination of immaturity of the immune system and a lower degree of priming. Antibody responses among the elderly are also generally reduced (89,98,99), possibly as a result of immune senescence and increasing prevalence of underlying diseases (100). Senescence of immune responses with age has been attributed primarily to loss or alteration of T-cell function (101). It has been suggested that under some circumstances, measurement of the cellular response to vaccination is a better predictor of subsequent protection in the elderly than is the measurement of antibody responses (102). Several groups of adults with potentially decreased responses to inactivated influenza vaccine have been identified, including those with renal disease (103) and transplant

Table 2 Effect of Strain Change and Dosing Interval on Serum Antibody Responses in Immunologically Naı¨ve Children Receiving Two Doses of Inactivated Influenza Vaccine Antigen

Dose 1

Dose 2

Two seasons with no change in formulation (94) H1N1 A/New Caledonia/99 A/New Caledonia/99 H3N2

A/Panama/99

A/Panama/99

B

B/Hong Kong/02

B/Hong Kong/02

Two seasons in which the formulation changed H1N1 A/New Caledonia/99 A/New Caledonia/99 H3N2 B

A/Wyoming/03 A/Panama/99 B/Jiangsu/03 B/Hong Kong/02

Source: From Ref. 95.

A/Wyoming/03 A/Wyoming/03 B/Jiangsu/03 B/Jiangsu/03

Dosing schedule

HAI test antigen

Fall, fall Spring, fall Fall, fall Spring, fall Fall, fall Spring, fall

A/New Caledonia/99

Fall, fall Spring, fall Fall, fall Spring, fall Fall, fall Spring, fall

A/Panama/99 B/Hong Kong/02

A/New Caldonia/99 A/Wyoming/03 B/Jiangsu/03

% response

Postvaccination GMT

78 76 89 88 51.7 59.8

47.7  3.1 57.2  4.2 114.6  3.3 129  3.7 24.3  3.9 28.1  3.9

93 86 83 70 88 39

91.9 79.5 77.8 57.1 61.6 18.0

     

2.6 3.3 3.7 4.1 2.5 2.4

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recipients (104–106). The responsiveness to influenza vaccination in HIV-infected individuals is related to the degree of immunosuppression (107,108). Most patients with chronic lung disease respond reasonably well to vaccination, and steroids at doses commonly used to treat reactive airways disease do not appear to preclude vaccine responses (109,110). Efficacy and Effectiveness In the years since the first introduction of inactivated influenza vaccines into clinical practice, there have been many studies devoted to assessing the potential benefits of vaccination, conducted in healthy adults as well as in children, the elderly, and other high-risk populations. In general, two types of end points have been evaluated: those in which the effect of vaccination on the rates of illnesses shown to be directly attibutable to influenza by laboratory testing are evaluated, and those which assess the effect of vaccination on illnesses assumed to be influenza related but in which no laboratory confirmation is attempted. For purposes of this discussion, we will refer to the former type of study as an efficacy evaluation, and the latter as an effectiveness evaluation. Protection against effectiveness end points will be diluted to the extent that there are non-influenza-related causes of the end point, which could not be reasonably expected to be impacted by vaccine. Overall, benefits of vaccination have been consistently demonstrated across all age groups, although as expected, there are significant differences in the magnitude of the effect among studies in different populations and with differing designs. Healthy adults Inactivated influenza vaccine has been shown to be effective in the prevention of influenza A in controlled studies conducted in young adults, with levels of protection of 70% to 90% when there is a good antigenic match between vaccine and epidemic viruses (27,111,112). For example, in a placebo-controlled study in healthy adults (113), the efficacy of TIV for preventing culture proven influenza A illness in adults was 76% (95% CI 58–87%) for H1N1 and 74% (95% CI 52–86%) for H3N2. In another recent study, vaccine efficacy was 76% (114). Randomized trials of vaccine in adults have also assessed effectiveness end points. For example, vaccination of healthy adults is associated with decreased absenteeism from work or school, decreased numbers of physician visits, and overall antibiotic use (115,116). Immunization of HCWs is effective in reducing days of absence from work and febrile respiratory illness rates (117,118). Children Few randomized controlled trials assessing inactivated influenza vaccine efficacy in healthy children are available. In one such study, the efficacies of inactivated vaccine were 91% and 77% in preventing symptomatic, culture-positive influenza A H1N1 and A H3N2 illness, respectively, compared with placebo (119). Immunization of asthmatic children has also been shown to reduce the incidence of influenza (120). A recent case-controlled study has also shown a significant reduction in laboratory-documented influenza among fully vaccinated children (121). Effectiveness studies of influenza vaccine have also confirmed the benefit of inactivated influenza vaccine in the pediatric population. Immunization of infants and children has been shown to protect against medically attended acute respiratory illness, influenza-like illness, pneumonia and influenza (P&I), acute otitis media (AOM), school absenteeism, work days lost by parents, and/or antibiotic use during influ-

enza epidemics (122–125). Vaccination of asthmatic children may shorten somewhat the duration of influenza-related asthma exacerbations (126). Elderly and high-risk groups Because it is generally believed that influenza vaccine is effective in elderly adults who are at higher risk for influenza-related complications, true placebo-controlled randomized trials of vaccine efficacy have rarely been conducted. In the most commonly referenced modern study, inactivated vaccine was approximately 58% effective in preventing serologically documented influenza in a population of adults over the age of 60 (127). However, the efficacy in the smaller subgroup of subjects 70 years of age and older was only 29%, and was not statistically significant. In another randomized trial, influenza vaccination was associated with a reduced risk of death and ischemic events among patients who were vaccinated after experiencing a myocardial infarction or angioplasty (128). Immunization with TIV was effective for prevention of influenza-related respiratory illness in another placebo-controlled trial among patients with chronic obstructive pulmonary disease (129). Vaccine has also been shown to be protective in limited studies in other high-risk groups, including those with HIV infection (130). A larger body of data exists from non-randomized or observational studies of vaccine effectiveness. These studies have suggested that influenza vaccination can reduce P&I hospitalizations and death among the elderly regardless of whether they have other conditions that place them at high risk for complications following influenza (5,131–136). In the largest recent evaluation, data from three health maintenance organizations over 10 influenza seasons were analyzed. Vaccination was associated with an overall 28% reduction in P&Irelated hospitalizations, and a 48% reduction in all-cause mortality in community-dwelling adults over 65 (137). Immunization confers significant health benefits among groups of elderly persons, including chronic lung disease. Influenza immunization has also been associated with reductions in the risk of hospitalization for heart and cerebrovascular disease, and the risk of primary cardiac arrest (138,139). Among nursing home residents, influenza immunization reduced the risk of hospitalization and pneumonia (140), and reduced the likelihood of influenza-like illness (141). In addition, vaccination is cost-effective, and can be associated with direct medical care savings (116,142,143). Because studies of this type are not randomized, there is the potential for introducing bias into the estimates of effectiveness. For example, physicians might be more likely to vaccinate individuals they consider to be at high risk for influenza complications, which might bias estimates away from efficacy since the vaccinated population would be enriched for high-risk individuals. Because the presence of these conditions is also contained in the medical record, studies that use health care databases are usually able to control for this type of bias using statistical techniques. Of greater concern is the impact of factors that are not readily available in the medical record (144). The healthy user effect (145) refers to factors that both increase the likelihood of vaccination and decrease the risk of disease in the vaccinated group independently of any direct benefit of the vaccine. For example, individuals who maintain a generally healthy lifestyle, and are relatively more compliant with other health care recommendations such as blood pressure control, diet, and exercise, or other medications, might both be more likely to seek and receive vaccine and less likely to experience an influenza

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Figure 2 New influenza antigenic variants can be rapidly attenuated by genetic reassortment with a well-characterized master attenuated vaccine virus. The attenuated vaccine master virus donates the gene segments encoding attenuation, while the epidemic virus donates the genes encoding the antigenically variant HA and NA in the construction of a live attenuated reassortant vaccine virus.

complication, stroke, or heart attack, or other morbidity event. Conversely, an individual may be too frail to travel to the clinic to receive vaccine, and thus be both less likely to be vaccinated and more likely to have a morbid event, increasing the risk in the non-vaccinated group. Revaccination The continued efficacy of repeated annual immunization has been questioned (146,147). Recent clinical trials have demonstrated that there are no consistent differences in postvaccination titers or proportion of subjects with putative protective titers when compared with subjects immunized for the first time, and that there is no consistent decrease or increase in the level of protection against influenza when multiple vaccination groups are compared with single vaccination groups (148–150). Revaccination of elderly persons was associated with a reduced mortality risk in one study (151).

Live, Attenuated (Cold-Adapted) Influenza Vaccines The use of live attenuated viruses as influenza vaccines offers several potential advantages over parenteral inactivated vaccines, including induction of a mucosal immune response, which more closely mimics that induced by natural influenza virus infection. In addition, the potential superiority of such vaccines in protection of the upper respiratory tract might be useful in strategies using vaccine to limit transmission of influenza. A key requirement for the development of attenuated influenza vaccines is the ability to rapidly attenuate new antigenic variants. The most widely used approach takes advantage of the segmented nature of the influenza virus genome to generate reassortant viruses in which the gene segments encoding attenuation are derived from a well-characterized master

donor vaccine virus, and the gene segments encoding the HA and NA are derived from the new antigenic variant (152,153) (Fig. 2). The currently licensed CAIV, utilizes the cold-adapted influenza A/Ann Arbor/6/60 (H2N2) and B/Ann Arbor 1/66 master donor viruses (154). The process of cold adaptation refers to the repetitive passage of a virus at gradually decreasing temperature until a virus is isolated, which replicates efficiently at a low temperature at which the replication of the original wild-type virus is significantly restricted. During this process, additional mutant phenotypes are frequently acquired. The cold-adapted influenza viruses demonstrate three such phenotypes: (i) the cold-adapted (ca) phenotype, defined as the ability to replicate efficiently at 258C, a restrictive temperature for wild-type influenza viruses; (ii) the temperature-sensitive (ts) phenotype, defined as significant (>2 log10) restriction of virus replication at 388C to 398C; and (iii) the attenuation (att) phenotype, defined as restricted replication in the lower respiratory tract of experimental animals (155). The genetic basis of attenuation of the master donor viruses has been determined by a combination of sequence analysis, traditional assessment of genetic reassortant viruses, and reverse genetics techniques. Multiple mutations appear to be involved in the attenuation of both donor viruses (Table 3). In the case of the ca A/Ann Arbor/6/60 virus, multiple mutations have been shown in all six of the so-called internal or non-HA or NA gene segments (155,156). Studies using single-gene reassortants have shown that at least three of these gene segments (PB1, PB2, and PA) participate in the attenuation of the ca influenza A virus in both animals and healthy seronegative human subjects (157,158). When individual mutations were placed in the genome using reverse genetics techniques, the ts phenotype of

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Table 3 Contributions of Individual Mutations to the ca, ts, and att Phenotypes of the Master Donor Cold-Adapted Viruses A/Ann Arbor/6/60 and B/Ann Arbor/1/66 Phenotypes Gene segment A/Ann Arbor/6/69 (ca, ts, att) PB2 PB1

PA NP M2 NS1 B/Ann Arbor/1/66 (ca, ts, att) PB2 PB1 PA NP

M

Mutations

ts

N265S

C

K391E

C

E457D E581G A661T K613E L715P T23N D34G A86S A153T S630N I651V V431M Y497H T55A V114A P410H A509T H159Q M183V

ca

att

Comment Mutation contributes to attenuation in ferrets. PB2 singlegene reassortant is attenuated in human. An engineered virus with all three mutations is attenuated in ferrets. PB1 single-gene reassortant is attenuated in human.

C C PA single-gene reassortant was attenuated in human.

C

C

C

C

C C

C C

C C C C

Abbreviations: ca, cold-adapted; ts, temperature-sensitive; att, attenuation.

the ca influenza A/Ann Arbor/6/60 virus was mapped to five gene sites, three in PB1, one in PB2, and one in NP (159). The PA, NP, and PB2 gene segments all independently contribute to the ca phenotype of the ca B/Ann Arbor/1/66 virus (160). Unique mutations involved in the ts and ca phenotype include two sites in NP and one in PA. These changes plus two additional changes in M1 are involved in the att phenotype as assessed in the ferret model (161). These findings are consistent with analysis of laboratory-derived revertant viruses also implicating the PA gene segment as playing an important role in attenuation (162,163). Two important consequences of the genetic basis of attenuation of these viruses should be noted (155). First, the alterations induced by the mutations in internal gene segments do not appear to be influenced significantly by the HA and NA. Therefore, the level of attenuation of both the ca influenza A and B viruses are reproducible from reassortant to reassortant, so that repeated clinical trials are not needed each time a new antigenic variant vaccine is generated. Second, attenuation is the consequence of multiple mutations on multiple gene segments. This means that the att phenotype is likely to be stable even after replication in seronegative children (164), and that there is little likelihood that reassortment events between vaccine and wild-type viruses would generate virulent viruses. Safety Cold-adapted influenza A and B viruses have consistently been extremely well tolerated in healthy adults, even among those with low levels of prevaccination antibody. Nasal symptoms (runny nose, nasal congestion, or coryza) and sore throat were the most frequently identified adverse symptoms attributable to vaccination in these studies (113,165–168). CAIV has also been well tolerated in adults with asthma or chronic obstructive airway disease (169,170). Vaccine is very well tolerated in the elderly,

although in one study, vaccine recipients had a 13% excess of sore throats compared with those who received placebo (165). Bivalent and trivalent CAIV have also been observed to be safe and well tolerated in children (119,171–175) with the most common symptoms following vaccine being runny nose or coryza, cough, headache, chills, vomiting, and abdominal pain. No significant vaccine-related adverse events have been seen in studies of children with cystic fibrosis (176,177), or asthma (178,179), and vaccinated children with asthma have not experienced significant changes in FEV1, use of b adrenergic rescue medications or asthma symptom scores compared with placebo recipients (179). However, in larger studies, wheezing has been consistently identified as a vaccine-associated side effect, although occuring at low rates (180). In the largest trial, medically significant wheezing within 42 days of vaccination was reported in 3.8% of children under two years after receipt of CAIV, compared with only 2.1% in those who received inactivated vaccine (181). Wheezing generally occurs in the youngest, not previously vaccinated children following the first dose of vaccine. Because of this observation, the coldadapted vaccine is currently licensed in the United States only for use in children two years and older, who do not have a history of asthma. Although CAIV is not intended for use in individuals with HIV, such persons could be vaccinated inadvertently. Small numbers of adults and children with relatively asymptomatic HIV infection have received CAIV (168,175) No excess or prolonged CAIV virus shedding was detected in HIVinfected compared with non-HIV-infected CAIV recipients. No significant changes in blood CD4 counts or quantitative HIV RNA levels after vaccination with CAIV were detected. Because CAIV is a live vaccine, there is the potential for transmission of vaccine virus to contacts. This has raised concern about the use of CAIV, particularly in some settings

Chapter 51: Influenza

such as hospitals. CAIV can be recovered from nasal secretions of about half of adult recipients, although generally, shedding of CAIV by adults is of low titer and short duration (182). Although young children shed much higher levels of vaccine virus, no transmission of CAIV from vaccine recipients to susceptible contacts was detected in studies of young children involved in day care–like settings, where CAIV and placebo recipients played together for up to eight hours a day for 7 to 10 days after vaccination (153,183). In the largest study, 197 children between 8 and 36 months of age were randomized to receive trivalent CAIV or placebo intranasally, and vaccine virus shedding was assessed for 21 days after vaccination. Although 80% of CAIV recipients shed at least one vaccine strain, for a mean of 7.6 days, transmission was detected in only one placebo recipient, for an estimated transmission rate of 1.75% (95% upper bound of 8%). Vaccine virus isolates recovered from vaccinated volunteer subjects have all retained the attenuated phenotype and genotype (184). Immune Response The mechanism of protection induced by cold-adapted vaccine has mostly been evaluated in experimental infection studies. Cold-adapted vaccine is protective in these experiments in the absence of significant serum HAI responses, suggesting that the main protective effect is induction of mucosal antibodies (185). However, protection can be demonstrated in some circumstances even in the absence of detectable mucosal responses (186), and the specific levels of mucosal antibody required for protection are unknown. In addition, the role that induction of cellular immune responses plays in the protective effect has been incompletely studied. Studies of the immunogenicity of cold-adapted reassortant vaccines have been carried out in children, adults, and elderly. The results of these studies are consistent with the hypothesis that the replication of cold-adapted vaccines in the upper respiratory tract, and hence their immunogenicity, is influenced by the susceptibility of the host at the time of vaccination. The frequency and magnitude of immune responses to vaccination is therefore highest in young children, intermediate in adults, and lowest in elderly subjects who have been repeatedly infected with influenza viruses throughout their lifetimes. In addition, the mucosally administered ca vaccine is generally more effective than parenterally administered inactivated influenza vaccine at inducing nasal HA-specific IgA, while inactivated vaccine usually induces higher titered serum HAI and HA-specific IgG antibody (187). Most susceptible children will demonstrate measurable serum and mucosal antibody responses (173,174,177,188,189). Post-CAIV-vaccination secretory antibody persists for up to or beyond a year in children. A study of trivalent CAIV administered to children 15 to 71 months of age demonstrated mucosal IgA strain–specific responses of 62% to 85% (190). In contrast, vaccination with bivalent or trivalent CAIV in unscreened adults generally results in a low proportion of more than four-fold rises in serum strain–specific influenza antibody (113,166,191) and relatively lower rates of mucosal responses (84). Even in those prescreened to have low prevaccination vaccine-specific influenza antibody, the rates of serum antibody responses to intranasal CAIV in adults and the elderly are low (166,192). B cell responses to both TIV and LAIV in infants, children, and adults have recently been reported (86). Influenza-specific IgA and IgG ASC peak on days 7 to 12 after either LAIV or TIV in both adults and older children, consistent with other studies

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in adults showing peak of ASC after TIV around days 7 to 8 (193–195). In children, there was no difference between TIV and LAIV, while in adults IgG ASC were significantly higher after TIV than LAIV. There was no difference between the two vaccines in children, and children had higher IgG ASC after LAIV than did adults. Generally, infants had the lowest amounts of ASC after TIV (LAIV not tested). Antibody responses were also significantly lower after LAIV than TIV in both adults and children. Development of ASC seemed to be a more sensitive indicator of take after LAIV than was antibody response. The levels of prevaccination memory B cells were low in all age groups, but numbers of prevaccination memory B cells were higher in adults than children. TIV, but not LAIV, increased the numbers of circulating memory B cells at one month. Influenza-specific interferon (IFN)- g-producing CD4þ and CD8þ lymphocytes have also been detected following both LAIV or TIV (196). In children five to nine years of age, TIV resulted in increases in the numbers of CD4þ but not in the numbers of CD8þ cells on day 10 following vaccination. There were no real changes in natural killer (NK) cells following TIV. In contrast, administration of LAIV resulted in increases in both IFN-g-producing CD4þ and CD8þ cells, as well as in NK cells. In adults, there were no consistent changes in any subset after either TIV or LAIV, although there was much variability in the responses. Efficacy and Effectiveness Children Trivalent CAIV was demonstrated to be efficacious in the prevention of influenza in a pivotal two-year, randomized placebo-controlled trial conducted in 1314 children aged 15 to 74 months (171). Efficacy against culture-confirmed influenza illness in the first year of this trial was 95% (95% CI 88–97%) against influenza A/H3N2 and 91% (95% CI 79–96%) for influenza B. In the second year of the trial, the H3 component of the vaccine (A/Wuhan/93) was not a close match with the predominant H3 virus that season, A/Sydney/95. However, the efficacy of CAIV against this variant was 86% (95% CI 75– 92%) (172). Overall, the efficacy of intranasal trivalent CAIV to prevent any influenza illness during the two-year period of surveillance in this field study was 92% (95% CI 88–94%). Studies done in Asia have reached similar conclusions, with an efficacy of CAIV compared with placebo of between 64% and 84% over multiple seasons, depending on the antigenic match with the vaccine (197). Studies conducted in healthy young children have generally concluded that trivalent CAIV may be more efficacious than TIV, including both against viruses which are well matched antigenically to the vaccine virus and those which are antigenically drifted (Table 4) (181,198,199). The enhanced efficacy in children determined in these trials is consistent with the results of earlier trials using monovalent or bivalent vaccines (119). Adults The currently licensed trivalent CAIV was approved in adults on the basis of the results of a large effectiveness trial performed in 4561 healthy working adults (167). In this study, the effectiveness of trivalent CAIV in preventing severe febrile respiratory illness of any cause during the influenza season was 29%. In subsequent studies, the protective efficacy of trivalent CAIV against laboratoryconfirmed influenza in healthy adults has been demonstrated to be approximately equivalent to that provided by inactivated vaccine (Table 4). In a recent three–armed study, the efficacy of trivalent cold-adapted vaccine compared with placebo for prevention of laboratory-confirmed influenza in healthy adults was 57%, while the efficacy of the inactivated vaccine was 77%,

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Table 4 Comparative Studies of the Protective Efficacy of TIV and CAIV in Adults and Children Study

Year

Population

End point

Virus

Groups

Incidence rate (%)

Protective efficacy (95% CI)

181

04–05

Healthy children 6–59 mo

Culture-confirmed illness

H1N1

TIV CAIV

0.7 0.1

a

H3N2

TIV CAIV TIV CAIV

4.5 0.9 3.5 2.9

a

Placebo TIV

7.1 0.61

a

CAIV Placebo TIV CAIV

0.32 4.3 1.0 1.4

H1N1

TIV CAIV

1.0 0

a

H3N2

TIV CAIV TIV CAIV

1.2 1.7 3.7 1.1

a

H1N1

TIV CAIV

0.5 0.0

a

H3N2

TIV CAIV TIV CAIV

1.2 1.5 5.0 3.2

a

Placebo TIV

2.9 0.8

a

CAIV Placebo TIV CAIV

0.8 2.9 0.6 1.7

B

119

85–90

Children <16

Culture-confirmed illness

H1N1

H3N2

199

02–03

Children 6–71 mo

Culture-confirmed illness

B

198

02–03

Children 6–17 yr with asthma hx

Culture-confirmed illness

B

114

04–05

Healthy adults 18–46 yr

Culture-confirmed illness

H3N2

B

113

85–90

Adults and children

Culture-confirmed illness

H1N1

H3N2

Placebo TIV CAIV Placebo TIV CAIV

89.2 (67.7, 97.4) 79.2 (70.6, 85.7) a

16.1 (7.7, 34.7)

91.4 (63.8, 98.0) 95.5 (66.7, 99.4) a

77.3 (20.3, 93.5) 67.7 (1.1, 89.5)

100 (56.0, 100.0) 47.9 (236.5, 32.6)

a

68.9 (39.2, 85.2)

100 (15.6, 100.0) 29.9 (190.9, 40.6)

a

36.8 (1.6, 59.8)

74 (11, 95) 74 (12, 95) a

80 (8, 97) 40 (103, 81) a

76 5/8 87 85 70 92 a

74 52 86 58 29 75

Comparison group for calculation of protective efficacy. Abbreviations: CAIV, cold-adapted influenza vaccine; TIV, trivalent inactivated vaccine.

a

but the difference between the two vaccines was not statistically significant (114). These results are consistent with those of an older trial using bivalent CAIV (not containing an influenza B component) (113). Finally, in the human challenge model, coldadapted and inactivated influenza vaccines were of approximately equal efficacy in prevention of experimentally induced influenza A (H1N1), A (H3N2) and B (166). The combined efficacy in preventing laboratory-documented influenza illness due to the three wild-type influenza strains was 85% for the CAIV and 71% for the inactivated vaccine. Elderly and high risk No studies of the protective efficacy of ca vaccine alone have been conducted in the elderly, because of the possibly reduced immunogenicity of the vaccine in this age group. However, the combination of local live attenuated influenza vaccine and parenteral inactivated vaccine administered together was shown to result in an approximately 60% decrease in cases of laboratory-confirmed influenza in an elderly nursing home population, compared with inactivated vaccine alone (200). However, this protective effect of combined vaccination could not be demonstrated in a population of adults with chronic obstructive pulmonary disease (201).

OPPORTUNITIES AND NEEDS FOR IMPROVED INFLUENZA VACCINATION The two currently available approaches to vaccination against seasonal influenza are effective interventions that can significantly reduce the impact of a public health threat. Yet, there are several areas in which it is generally recognized that the perfomance of the current vaccines could be improved. The production of both types of the vaccine in embryonated hen’s eggs is problematic for several reasons, and development of cell culture production is needed. There are significant concerns regarding the protective efficacy of influenza vaccine in elderly and debilitated populations, and methods to enhance immunogenicity are also a high priority. The world’s production capacity is likely to remain limited in relation to the potential demand, and methods to expand the supply either by increasing production or exploring dose-sparing strategies, are important to explore. The protection afforded by current vaccines is narrowly focused on the specific strains included in the vaccine, but it is generally recognized that vaccines that would provide broad and durable cross-protection among strains within a subtype, or even across subtypes, would be highly desirable. The growing potential pandemic threat posed by

Chapter 51: Influenza

recent human infections with pandemic viruses has magnified the need for better vaccination strategies, and development of more effective strategies for pandemic prevention may have important implications for seasonal influenza as well. Although the current vaccine is very well tolerated, concern about side effects is one factor that is frequently cited as a reason why individuals do not choose to receive influenza vaccine. In addition, successful implementation of vaccine strategies will necessarily require exposure of large numbers of recipients to potential side effects. Therefore, any new influenza vaccine must be very safe, and any significant increase in local or systemic reactogenicity over that seen with current vaccines would probably be unacceptable.

Vaccines Produced in Alternative Substrates Routine production of inactivated influenza vaccines in embryonated hen’s eggs has several practical drawbacks, including the expense and difficulty in ensuring the availability of sufficient numbers of eggs in the compressed time frame in which influenza vaccine is prepared, and the potential vulnerability of flocks to avian diseases. Eggs are also susceptible to contamination with bacteria. An additional concern is the potential that antigenically variant influenza A viruses can be selected during the process of developing strains for vaccine production, which grow well in eggs. Direct PCR amplification of viruses in nasal secretions has documented that the HA of the major population of virus shed from the nasopharynx of infected humans is identical to that isolated in Madin-Darby canine kidney (MDCK) cells, and significantly different from that isolated in eggs. Antigenic differences of this type have been documented for H3N2, H1N1, and influenza B viruses (202–209). Alterations in CTL epitopes have also been described (210). Although these differences may amount to only a few amino acids, the MDCK cell–grown virus appears to be more effective than egg-grown virus as an inactivated vaccine for protection of experimental animals (211,212). Thus, there is considerable interest in developing alternative, cell culture–based substrates for production of influenza vaccines. For these reasons, the use of mammalian cell culture for growth of influenza viruses for vaccine production is being explored. The main technical challenge has been to develop mammalian cell lines suitable for use as vaccine production substrates (213), and to optimize culture techniques so as to obtain high yields. Three cell lines, MDCK, a canine epithelial cell line, Vero, an epithelial cell line derived from monkey kidney, and Per.C.6, an adenovirus-transformed human conjunctival cell line, have received the most attention (214). Studies conducted in humans have shown that the immunogenicity evaluation of a candidate MDCK cell–grown vaccine generated antibody titers similar to those generated by egggrown vaccines at similar doses (215,216). Use of recombinant DNA techniques to generate vaccine antigen expressed in cell culture is another alternative that also allows control over the sequence of the HA used. Recombinant HA antigens generated in insect cells by recombinant baculoviruses have been evaluated extensively in healthy adult and elderly populations as both monovalent and multivalent preparations (217–219). The vaccines elicit functional antibody responses, including both HAI and neutralizing antibody, in a dose-dependent manner, and at the higher doses evaluated (up to 135 mg per component) antibody responses surpass those seen following conventional subvirion vaccine in elderly adults (220). In a recent trial, a formulation containing 45 mg per HA

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component was shown to provide 86% protective efficacy against culture-confirmed influenza (221). These results suggest that the known differences in glycosylation in insect as compared with mammalian cells (222) do not affect the generation of a protective antibody response in primed individuals, but studies in children have not been reported.

Neuraminidase Vaccines The infection-permissive nature of anti-NA immunity has stimulated interest in the use of NA-based vaccines. Such vaccines could provide clinically relevant protection, and yet allow subclinical infection with the development of immune responses due to virus infection. Studies of purified NA antigens have demonstrated that they induce NA antibody effectively, and can prevent death in lethal mouse models of influenza (34,223–225). Because the NA appears to undergo antigenic drift at a slower rate than the HA, an NA or NAsupplemented inactivated vaccine may induce better protection against drifted viruses than does standard inactivated vaccine (226). Purified NA vaccines have been developed, and clinical trials have demonstrated dose-related increases in NA-specific antibody (227). Preliminary results in the human challenge model have suggested that an NA-supplemented vaccine may be more efficacious in protection against an antigenic variant than was standard vaccine (228). Supplementation of TIV with additional NA is also an option.

High- or Low-Dose Vaccine Studies conducted over many years have established that increasing doses of HA antigen are associated with increased antibody responses, although the dose response curve is rather flat. Thus, if an increased dose was found to be feasible to produce, it could represent a simple approach to enhanced immunogenicity, particularly in the elderly. Studies using purified HA have demonstrated increases in serum and nasal secretion antibodies with increasing doses in the range of 15 to 405 mg HA antigen being given to young adults and 15 to 135 mg to elderly persons (83,229). Similarly, the serum HAI antibody response among healthy elderly subjects was significantly greater following trivalent vaccine at 60 mg per HA than after the standard dose of 15 mg per HA (230). Although dose-related increases in injection site reactions were noted, vaccines were well tolerated at all dosage levels. Healthy adults also respond well to lower doses of vaccine. In one large randomized trial, (231) the serum antibody response of healthy adults to 0.25 mL (7.5 mg of each HA antigen) of trivalent inactivated influenza vaccine was only slightly lower than the response to a 0.5 mL dose (15 mg of each HA antigen). The proportions of individuals achieving a protective titer of 1:40, the geometric mean titers of postvaccination antibody, and the proportions of individuals with a fourfold or greater increase in antibody were lower for all three strains in those receiving 0.25 mL of vaccine. However, the differences were small. These results suggest that in situations of vaccine shortage, a strategy using a half dose in healthy adults could increase the number of people vaccinated with relatively little impact on protective efficacy. In addition, in a small, randomized trial, there was little or no difference in the effectiveness of a half dose compared with a full dose of vaccine in preventing influenza-like illness among healthy adults (232). These findings are consistent with the conclusion of an extensive meta-analysis of previous studies, which concluded that an

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Figure 3 Emusions used as adjuvants with influenza vaccine may be either water in oil or oil in water. Source: From Ref. 241.

increase in dosage from 10 mg to 15 mg would not be associated with a substantial increase in protection in healthy adults (233).

Intradermal and Transcutaneous Immunization Because of the high concentration of antigen-presenting cells in the skin, this organ is an attractive site for immunization. Doses of 0.1 mL administered intradermally (ID) have been reported to be as immunogenic or more immunogenic as a 0.5 to 1.0 mL dose given intramuscularly (IM) or subcutaneously in some studies (234), but not others (235). In children, the 0.1 mL dose also appeared to be as immunogenic as a full dose given IM (236). However, it is important to bear in mind the relatively flat dose response to influenza vaccine noted above when interpreting these studies. When the same doses are compared side-by-side, the ID route was not superior to IM vaccine at inducing antibodies. In addition, ID vaccine induced significantly more local inflammatory response than IM vaccine (237). If the skin is treated in such a way as to remove or alter the barrier function of the stratum corneum, antigen can penetrate to the layer of the epidermis that is rich in Langerhans cells (a form of dendritic cells). In the transcutaneous immunization approach, antigen is applied directly to hydrated skin, in the form of a patch, for several hours. When the stratum corneum is hydrated, antigens can penetrate the skin and become available to antigen-presenting cells (238). A second approach involves application of the antigen to the skin using a powder vehicle such as trehalose applied with the gene gun device (239). Both systemic antibody and CTL responses have been reported, and remarkably, mucosal immune responses as well. Recently, an approach based on cyanoacrylate skin surface stripping has also been reported to induce antibody as well as effector CD4 and CD8 T-cell responses to vaccine (240).

Adjuvants and Alternative Formulations The potential for adjuvants to enhance antibody responses to influenza vaccines has been explored since the early years of their development. Generally, adjuvants have multiple mechanisms of action, including irritation at the site of injection, creation of a depot effect, formation of particles that improve

presentation to antigen-presenting cells, and the engagement of toll-like receptors (TLRs) (241). Many adjuvants use multiple mechanisms to enhance immune responses or combinations of adjuvants to maximize immune responses. Emulsions (Water in Oil, Oil in Water) Both water-in-oil and oil-in-water emulsions have been evaluated as adjuvants for influenza vaccine in man (Fig. 3) (241). Significant increases in serum antibody titers were observed in subjects in the United States and Britain given influenza vaccine in water-in-oil, mineral oil adjuvant in the 1950s, and their use in the American military conferred significant protection against clinical influenza (242). The development of abscesses at the injection site led to discontinuation of the use of these vaccines. In contrast, the oil-in-water emulsion MF59 was well tolerated and resulted in slightly higher GMT rises than seen with standard subunit vaccine in healthy adults (243,244). This vaccine has also been well tolerated and induced somewhat higher titered serum antibody responses in the elderly than does standard splitproduct vaccine (245,246). In chronically ill, older adults, use of MF59 was associated with enhanced immunity (247) and improved responses to antigenic variants (248). Importantly, no induction of antibody against squalene has been noted (249). In an effectiveness trial conducted in long-term care institutions, the relative risk of influenza-like illness was reduced in subjects with preexisting chronic respiratory disease or cardiovascular disease receiving MF-59 adjuvanted vaccine (250). Toll-like Receptor Agonists Direct stimulation of TLRs by associated agonists has emerged as an effective means of stimulation of the innate immune system, and potentially enhances the adaptive immune response. The oligodeoxynucleotide CpG is a potent agonist for TLR-9, found on B cells and plasmacytoid dendritic cells in humans. However, CpG failed to enhance serum antibody responses to influenza vaccines in healthy younger adults (251). Cytokines and Chemokines Several cytokines and immunomodulators have been evaluated for their ability to enhance influenza vaccine immunogenicity.

Chapter 51: Influenza

Reports of enhanced antibody responses among elderly persons given thymosin-a1 and interleukin-2 suggest that addition of an immunomodulator may benefit selected populations (252–254). Dihydroepiandosterone (DHEA) has also been reported to have a modest enhancing effect (255). However, the growth factor GM-CSF failed to enhance serum antibody responses in healthy younger adults (256). Formulations, Combination Adjuvants Attempts to physically modify the vaccine to improve antigen presentation, such as formulation in liposomes (257,258), or multimeric complexes such as ISCOMs (immunostimulating complexes) (259,260). A liposomal vaccine formulated with IL-2 was more immunogenic than TIV among elderly subjects (261). Formulation of HA into ISCOMS with the adjuvant Quil A has resulted in improved antibody and CTL responses in phase I studies in humans (262,263). QS21, a saponin derived from Quil A, failed to augment antibody or cell-mediated responses in healthy adults when given with TIV, compared with TIV alone (264). Virosomes (virus-like particles of 100–150 m in diameter containing HA within the membrane) are approved for use in Europe, and are reportedly more immunogenic than standard inactivated vaccines (265). Structurally, virosomes are spherical vesicles of approximately 150 nm in diameter, composed of a lipid membrane with integrated envelope proteins derived from influenza virus, predominantly HA (266). Because virosomes retain the cell binding and membrane fusion properties of the native virus, they are thought to interact efficiently with antigen-presenting cells, resulting in activation of T lymphocytes (267). In a recent study, a virosomal vaccine had similar immunogenicity as MF-59 adjuvanted vaccine in elderly, but lower rates of local side effects (268). Virosomal vaccine was also well tolerated and immunogenic in young children (269).

DNA Vaccines Immunization of mice with DNA encoding the HA, as well as the internal M and NP proteins of influenza A, induces longlived humoral and cellular immune responses (270,271), which are protective against viral infection and disease. Immunization of African green monkeys with DNA encoding a combination of three HAs and other influenza virus genes induced serum antibody against all three HAs (272). Most studies in humans of DNA vaccination for influenza have not shown impressive responses. However, epidermal delivery of DNA in the form of gold particles has been reported to elicit strong HAI antibody responses in humans (273).

Strategies to Enhance or Broaden Immune Responses to Other Viral Proteins While the first priority for enhancing vaccine immune response is to increase antibody levels to the HA, optimizing responses to other viral proteins should improve protection against influenza. Because several of these epitopes (M, NP) are conserved between influenza A subtypes, vaccines based on these proteins offer the potential for increasing the breadth and duration of protection against diverse subtypes. Immunization with purified M2 protein has also been shown to ameliorate infections in animals (274), and vaccines based on the external domain of the transmembrane M2 protein, M2e, elicit crossprotective antibodies in a murine model (275,276). Because the

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immunity is less potent than HA-based immunity, and escape mutants emerge, it is likely that M2e will be used as an adjunct to current vaccines rather than as a stand alone antigen (277). Additional conserved determinants for protective antibodies are likely to exist (278). One such potentially cross-reactive epitope is the highly conserved maturational cleavage site of the HA(0) precursor of the influenza B virus HA, which has been shown to elicit a protective immune response to nonantigenically cross-reactive influenza B virus lineages (279). CTLs are the principal mediators of recovery from pneumonia in the mouse model of influenza (280). CTLs likely are also important in hastening recovery and preventing pneumonia in humans. The main CTL targets in humans are conserved epitopes on the NP and M1 proteins (90). Recombinant DNAexpressed NP protein, plasmid DNA, and recombinant adenoviral vaccines induce CD8 CTL responses in mice that mediate protection against severe disease (270,281,282). Use of NP and M1 vaccines (purified proteins or plasmid DNA constructs) to expand memory cell populations for CTLs is under consideration.

Intranasal Inactivated Influenza Vaccines Studies in humans conducted over many years have shown that nasopharyngeal administration of inactivated vaccines by nose drops or aerosol can stimulate production of local antibody in primed individuals (283–287). However, the simple administration of soluble antigen to this mucosal surface is inefficient, requiring relatively large amounts of antigen to induce mucosal immune responses. Research has therefore focused on ways to enhance the immunogenicity of mucosal inactivated vaccine by increasing uptake of antigen by mucosal antigen-presenting cells. Strategies used have included mucosal adjuvants, incorporation of HA and other antigens into particulate formulations, or both. Bacterial enterotoxins, such as cholera toxin (CT), have been extensively evaluated as mucosal adjuvants for influenza and other vaccines. However, these toxins are far too reactogenic in man for routine use mucosally, as microgram quantities can induce cholera diarrhea if they reach the small intestine. Initial studies showing a potential adjuvant effect of purified B subunit (288,289) were complicated by the presence of residual amounts of holotoxin (290), and it became clearer that the holotoxin was responsible for the majority of the adjuvant effect (291). Further development has focused on engineering mutations designed to reduce or eliminate the diarrheagenic potential of CT or the highly related heat-labile toxin (LT) of enterotoxigenic Escherichia coli, while retaining adjuvanticity. Two types of mutations have shown promise: mutations that block the enzymatically active (ADP-ribosylating) site (e.g., LTK63 or LTR172) and mutations that block the protease activation site (e.g., LTG192) (292). The largest experience in humans with intranasal inactivated influenza vaccine has been with HA formulated in phosphatidylcholine liposomes, and administered with fully enzymatically active LT. This vaccine was well tolerated in adults, children, and the elderly, and induced strong nasal antiHA IgA responses in adults (293). Responses were less strong in the elderly, but the vaccine exceeded European licensing guidelines for serum antibody in all age groups (294). In addition, the vaccine was reported to be protective in adults and children, and to reduce the frequency of otitis media in otitis-prone children (295,296). However, this vaccine was later

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withdrawn from use after multiple reports describing the onset of Bell’s palsy shortly after vaccine administration (297). These observations were consistent with studies in rodents showing that the strong avidity of enterotoxins for GM1 gangliosides present on neuronal cells of the olfactory bulb can result in colocalization of toxin and antigen in neural tissue (298). Thus, intranasal administration of enterotoxins could result in low level neural inflammatory responses. A number of additional adjuvants or alternative formulations that do not include enterotoxins have been evaluated in humans with inactivated influenza vaccine for intranasal immunization. These include use of liposomes (299), and the MF59 oil-in-water adjuvant intranasally (300). Intranasal administration of purified HA and NA in proteosomes (mixtures of influenza HA and outer membrane protein vesicles of group B Neisseria meningitidis) has resulted in modest serum HAI responses, with detectable nasal IgA responses in 50% to 70% of subjects (301–303). A small field efficacy study of a trivalent proteosome intranasal vaccine has been reported to show statistically significant efficacy in prevention of laboratory-documented influenza illness.

Other Live Viral Vaccines The development of techniques for direct genetic engineering of specific mutations in the genome of influenza A and B viruses (Fig. 4) (305–307) has provided an important tool for generation of potential live influenza vaccine candidates. Directly introduced mutations at several sites have been shown to result in

attenuation. These include replacement of the 30 and 50 ends of a gene segment of influenza A virus by the corresponding regions of an influenza B virus (308), manipulation of the stalk of the NA (309,310), and placement of avian-like sequences into the PB2 gene (311,312). One intriguing approach has been complete or partial deletion of the NS1 protein (313). These viruses manifest significantly reduced replication in systems in which the type I interferon system is intact, implicating the NS1 protein as an interferon antagonist. Complete or partial NS1-deleted viruses provide excellent protection against homologous wild-type influenza virus in mice (314), and are being developed as possible second generation vaccines.

VACCINES FOR PANDEMIC INFLUENZA Vaccination against pandemic influenza poses a much different challenge than vaccination against seasonal influenza, because it is anticipated that in the case of a pandemic, the majority, if not all of the population, will be immunologically naı¨ve to the emergent subtype. In addition, pandemics are unpredictable, and the manufacturing surge required to supply pandemicspecific vaccine, which can only commence following characterization of the new virus, may be unable to fulfill demand. While the use of monovalent rather than trivalent vaccine would likely increase the supply, this would be offset by the need for a two-dose schedule and increased antigen content (315). Thus, the focus of pandemic vaccine development has been on the use of adjuvants and vaccine formulations that maximize immunogenicity while using less antigen.

Figure 4 Direct genetic manipulation of the influenza virus genome. Cells are simultaneously transfected with plasmids expressing each of the eight virion RNAs and with plasmids expressing, at a minimum, the viral polymerase (PA, PB1, and PB2) and nucleoproteins. Transfection of the plasmids shown in brackets is not required but increases the efficiency of recovery of transfectants. Source: From Ref. 304.

Chapter 51: Influenza

History The most recent experience in pandemic vaccine development occurred in the late 1970s. After an absence of almost 20 years, an outbreak of swine influenza (H1N1) in Fort Dix, New Jersey, in 1976 triggered the urgent development and clinical testing of egg-grown whole-virus, split or subunit A/New Jersey/8/76 (H1N1) vaccines (21,316–320). Subsequently, the actual emergence of epidemic A/USSR/77 (H1N1) viruses triggered additional evaluations of H1N1 vaccines. Several relevant observations were made in these studies. There were clear differences in response between those aged less than 24 years and who had not been previously exposed to H1N1, and those aged more than 24 years who presumably had been exposed to H1N1 viruses in circulation before 1957. In naı¨ve subjects, if one dose of vaccine was administered, antigen content in excess of 60 mg HA was required to induce satisfactory responses. However, if two vaccine doses were given, much lower antigen content (5 mg) was needed. Furthermore, whole-virus vaccine was significantly more immunogenic than subunit or split vaccines in naı¨ve subjects, while among primed subjects, there was no difference in immunogenicity between wholevirus and subunit or split vaccines. Whole-virus formulations were also associated with greater reactogenicity, particularly in children, who developed febrile reactions even after low doses (317). Finally, aluminum salts were not particularly effective when used as an adjuvant with subunit vaccine (318). The more recent focus of pandemic vaccine development has been avian influenza A viruses. Influenza A viruses are enzootic in migratory waterfowl. In these birds, viruses with at least 16 different HA and nine different NA subtypes have been identified. Genetic analysis of the 1918 H1N1, 1957 H2N2, and 1968 H3N2 pandemic influenza viruses of the 20th century is consistent with the hypothesis that new pandemic viruses derive their novel HAs and/or NAs from avian viruses, and emerge in man either by direct adaptation or by reassortment with human influenza viruses (321). For this reason, recent observations of severe human disease associated with avian H5N1 virus infections, first made in Hong Kong in 1997 (322), are of great concern. While effective vaccines against H5N1 are the most urgent need, avian H7 and H9 subtypes have also infected humans, and represent potential pandemic threats (322–324).

Vaccine Development for H5N1 Viruses Avian H5 and H7 viruses pose particular challenges for vaccine development because they may possess the ability to replicate systemically in birds. This feature is related to the presence of a series of basic amino acids at the protease cleavage site of some H5 and H7 HAs, which increases the range of proteases capable of cleaving the HA, and which is associated with enhanced virulence in birds, and possibly other species. Because of this enhanced virulence, vaccine production from highly pathogenic avian influenza viruses (HPAIV) requires heightened biocontainment to protect workers and the environment from contamination. Furthermore, HPAIV have greater lethality to embryonated hens’ eggs, the usual growth substrate for influenza vaccine viruses, and thus may have lower yields. After the 1997 H5N1 outbreak, approaches to overcome such issues were evaluated. The use of nonpathogenic H5 virus strains capable of inducing cross-reactive immunity to the wild-type H5N1 viruses was investigated. Inactivated whole-virus and subunit H5 vaccines protected against lethal H5N1 challenge in mice

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(325–327). Surface-antigen vaccines containing 7.5 to 30 mg H5 HA from A/duck/Singapore/97 (H5N3), with or without MF59 oil-in-emulsion adjuvant, were clinically evaluated (328,329). Non-adjuvanted vaccine was poorly immunogenic, with only a 36% response rate after two 30 mg doses. The addition of MF59 was striking, giving significantly higher antibody titers and a 94% response rate after two doses. Antibody titers to H5N1 were about half those to H5N3, demonstrating the need for close antigenic matching between vaccine and pandemic strains to ensure optimal vaccine efficacy. Another approach that aims to overcome the need for egg-derived vaccines, involves the production of baculovirusexpressed purified H5 HA protein. Baculovirus-derived H5 and H7 HA vaccines protect against lethal virus challenge in chickens (330). However, recombinant baculovirus H5 vaccine was modestly immunogenic in unprimed humans with only a 52% response rate after two doses of 90 mg (331). Since this original outbreak, systems for directly engineering the genome of influenza viruses, discussed above, have been developed. Using these systems, highly pathogenic influenza viruses can be manipulated to remove the polybasic amino acid sequence responsible for virulence, and reassorted with viruses that grow well in eggs (332). This technology is reliable for the manipulation of H5N1 viruses, and safe vaccine reference viruses can be generated within weeks, although experience with other subtypes is limited. Although antigen egg yields from some reverse genetic H5N1 viruses seem lower than that expected of seasonal influenza viruses, this technique has been quite useful for the generation of vaccine strains for pandemic and interpandemic influenza. Another consideration for H5N1 vaccine development is the antigenic diversity of circulating avian H5N1 viruses. At least two antigenically distinct clades of H5N1 viruses containing several diverse sublineages (333) are currently circulating. Clade 1 H5N1 viruses are predominant in parts of China and the Indochina peninsula. Isolates from Indonesia and other parts of China are clustered in a more divergent clade 2 group. Clade 2 viruses have spread west into Europe and Africa, and are currently responsible for the majority of human infections, and have undergone considerable antigenic evolution, with multiple subclades now described. In contrast, the clade 3 viruses that were responsible for the first human H5N1 infections in 1997 have not been isolated since that time. On the basis of the experience with seasonal vaccines and limited information from clinical trials of H5 candidate vaccines, subjects responding to subvirion clade 1 H5N1 vaccines exhibit no or minimal cross-reactive antibodies to clade 2 viruses. The ability to induce broad immune responses is a highly desirable property for a pandemic vaccine. Subvirion H5N1 Vaccines Because non-adjuvanted, subvirion-inactivated influenza vaccines are the most widely licensed and used form of influenza vaccination, there would be considerable regulatory advantages if the same process could be used to generate a pandemic vaccine. Therefore, initial studies have focused on vaccines made by licensed manufacturers using a process as similar as possible to that used for their seasonal vaccine, except that the reference virus is generated using reverse genetics techniques. However, for reasons that are not clear, such traditional subvirion vaccine preparations have been relatively poorly immunogenic in healthy young adults (334,335). For example, administration of two doses, each containing 90 mg, was

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required to achieve immune responses that met licensing criteria in the United States (334). Since this represents a total dose of HA that is approximately 12 times that used for seasonal vaccination, there is considerable concern about the implications of this type of dose requirement on vaccine supply. Among children aged two to nine years, two 45 mg doses of subvirion H5N1 vaccine were well tolerated, but modestly immunogenic giving a 59% response rate (336). In elderly (aged >65 years) recipients, the responses to subvirion H5N1 vaccine paralleled the poor immunogenicity observed in younger adults, with only 35% responding to two doses of 90 mg (336). ID administration of H5 HA has not improved immunogenicity (336). Whole-Virus H5N1 Vaccines Although in some studies in the late 1970s, whole-virus H1N1 vaccines were superior to subunit vaccines in H1N1 naı¨ve subjects, they were associated with increased febrile events, particularly in children. Consequently, many vaccine manufacturers globally have not maintained whole-virus production licenses or facilities, and for many manufacturers, the detergent disruption step is an important part of the inactivation process. Replacing large-scale split vaccine production lines with wholevirus vaccine production facilities will pose major regulatory, financial, and commercial challenges for many producers. However, whole-virion vaccines could be easier to produce, with less losses during the manufacturing process, and there have been a number of studies evaluating whole-virion vaccines as pandemic vaccine candidates. Cell culture–grown, whole-virus vaccines generated from clade 1 and 2 highly pathogenic H5N1 viruses have been produced in biocontainment. In mice they are immunogenic, induce cross-clade neutralizing antibodies, and protect against lethal challenge with H5N1 antigenic variants (337). In clinical trials, a cell culture, wild-type derived clade 1 H5N1 wholevirus vaccine with or without alum was strongly immunogenic, and induced clade 2 neutralizing antibodies in up to 50% of responders. The non-adjuvanted vaccines were more immunogenic than alum-containing comparator vaccines, with the best response observed after two doses of 7.5 mg vaccine. Egg-grown whole-virus inactivated vaccine candidates produced from reverse genetic H5N1 viruses also display improved immunogenicity over subvirion vaccines. In dose-ranging trials, wholevirus vaccines containing 6 to 15 mg H5 HA and alum-induced responses rates of up to 70% after one dose and 80% following a second injection (336,338,339). Adjuvanted H5N1 Vaccines Aluminum salts are generally not used as adjuvant in seasonal influenza vaccines as studies suggest little benefit (340). However, addition of alum to whole-virus H2 and H9 vaccines appeared to allow reductions in antigen content, although comparator vaccines without alum were not tested (341). Studies have reported on the use of aluminum salts (hydroxide or phosphate) with subvirion H5N1 vaccines (335,336). Overall, alum has shown little benefit, and does not enhance immunogenicity of subvirion H5 vaccines containing low antigen content. More promising results have been seen using emulsions. Traditional water-in-oil emulsions, generally mixtures of mineral oil and water using monooleate as an emulsifier, were highly effective adjuvants in man, but associated with significant local reactogenicity in studies done in the 1950s. However, oil-in-water emulsions based on the metabolizable oil, squa-

lene, have shown remarkable abilities to increase the response to H5N1 and H9N2 vaccines. The most completely studied is MF59, which is licensed in combination with seasonal influenza vaccines in some countries (discussed above). In studies with a clade 3 A/Duck/Singapore/97 H5N3 vaccine, the addition of MF59 oil-in-emulsion adjuvant significantly enhanced immune responses compared with non-adjuvanted vaccine (328). In addition, a third dose of adjuvanted vaccine boosted neutralizing antibodies that were cross-reactive to a range of clade 1 H5N1 antigenic variants (342). Experience with MF59adjuvanted split reverse genetic H5N1 vaccine in over 400 adults is consistent with these findings (336). Two doses containing 7.5 or 15 mg HA induced response rates between 77% and 83%. Two additional squalene-in-water emulsions have also been evaluated, AS03 and AF03. Studies in which AS03 was administered with a clade 1 H5N1 split-product vaccine containing 3.8 to 30 mg HA in subjects aged 18 to 60 years (343) showed that the adjuvanted formulations were significantly more immunogenic at all vaccine doses compared with nonadjuvanted vaccine comparators, and even with the lowest antigenic dose antibody titers reached levels expected to be associated with protection. Furthermore, over 75% of subjects developed neutralizing antibodies against an antigenically distinct clade 2 virus. Preliminary results with AF03 have shown similar results. All of these oil-in-emulsion adjuvants were associated with increased local reactogenicity, although the reactions were generally mild and self limiting, and were considered acceptable for a pandemic vaccine. Live Attenuated Virus Vaccines Against Avian Influenza While injectable influenza vaccines induce serum antibodies to influenza, they are poor at stimulating local secretory IgA in the respiratory tract. As mucosal IgA exhibits potential broader crossreactivity at the point of entry, live attenuated virus vaccines may offer wider protection against drifted antigenic variants that could be advantageous once a pandemic is underway. Concerns over the generation of a reassortant between a live virus vaccine containing an avian influenza virus and a co-infecting human strain may limit the use of such vaccines in the interpandemic period. Both H5N1 and H9N2 cold-adapted recombinant viruses are nonpathogenic, and protect against lethal challenge in mammalian and chicken models (344,345). Candidate live attenuated H5 vaccines have been evaluated for safety, replicative capacity, and immunogenicity in clinical studies (336). Two doses of coldadapted A/Leningrad/134/57 donor strain vaccine containing HA and NA from A/Potsdam/92 H5N2 reference strain were well tolerated and induced mucosal and systemic responses in 65% and 50% of recipients, respectively. Genotypically stable vaccine virus could be recovered in nasal samples for up to 11 days following immunization.

Vaccines Against Other Avian Influenza Viruses Inactivated vaccine candidates against avian H9N2 strains responsible for human infections have been produced. Subunit and whole-virus A/Hong Kong/1073/99 (H9N2, G1 lineage) vaccines were compared in U.K. healthy adults aged 18 to 60 years (346). Many subjects born before 1969 had preexisting antibody titers to H9N2. This was attributed to cross-reactivity acquired from exposure to an earlier influenza virus. In these subjects, one dose of either vaccine boosted anti-H9 responses to levels associated with protection. In contrast, in unprimed subjects born after 1969, two doses of vaccine were suboptimal,

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although whole-virus vaccine was significantly more immunogenic than subunit vaccine. In another study, alum-adjuvanted whole-virus A/Hong Kong/1073/99 vaccines containing 1.9 to 7.5 mg H9 HA were compared with non-adjuvanted 15 mg whole-virus vaccine (341). Two doses of any vaccine induced acceptable responses suggesting that alum allowed dose reductions while maintaining immunogenicity. Surface-antigen A/chicken/Hong Kong/97 (H9N2, G9 lineage) vaccine, with or without MF59, was evaluated among adults aged 18 to 30 years (347). The MF59-adjuvanted vaccine was strongly immunogenic at the lowest 3.75 mg dose, with immune responses induced after a single injection that were comparable with responses seen following two doses of non-adjuvanted vaccine. As H2N2 viruses circulated in humans between 1957 and 1968, people born after this period lack immunity and would be susceptible if H2 reemerged. Inactivated vaccines produced from A/Singapore/57 (H2N2) have been evaluated (341). Alum-adjuvanted whole-virus vaccines containing 1.9 to 7.5 mg H2 HA were compared with non-adjuvanted 15 mg whole-virus vaccine in young, immunologically naı¨ve adults. A single dose of any vaccine failed to induce a significant response; however a second dose boosted antibody titers to levels associated with protection in all vaccine groups, suggesting that significant reductions in antigen content could be achieved with the addition of alum.

Immunogenicity End Points and Licensing of Pandemic Vaccines New vaccines for seasonal influenza must induce protective immunity as measured by HAI tests and meet licensing criteria set out by the European Committee for Human Medicinal Products. However, standard HI tests are insensitive for the detection of antibodies to H5 and other avian strains, and alternative serological tests including modified (HI) and virus neutralization are needed (348,349). The lack of recognized correlates of immunity for these assays poses challenges to developing consistent immunological end points for clinical trials and vaccine registration. Moreover, the relevance of currently accepted standards of seroconversion for seasonal vaccines needs to be reconsidered for their relevance to pandemic vaccines (350). As considerable inter-laboratory variation exists for both HI and neutralization tests to seasonal influenza vaccine, comparison of vaccine immunogenicity trials conducted in different countries should be interpreted with caution until antibody standards can be developed (28).

Pre-pandemic Vaccination and Stockpiling To overcome likely shortfalls in supplies of pandemic-specific vaccine during the early pandemic period, stockpiling of prepandemic vaccine has been considered. Virological surveillance is essential to identify new variants and generate reference strains for distribution to vaccine producers as antigenic evolution of H5N1 and other avian viruses creates uncertainties over strain selection for vaccine production. Induction of neutralizing antibody that is cross-reactive to a range of virus variants or subtypes would be an important characteristic of an ideal pandemic vaccine. Oil-in-emulsion adjuvants and whole-virus H5N1 vaccines demonstrate superior immunogenicity over split vaccines, and also induce cross-clade neutralizing antibodies to H5 viruses, raising the possibility of developing vaccine strategies to prime sections of the population in

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advance of a pandemic. Recipients of a baculovirus-expressed recombinant HA vaccine derived from a clade 3 1997 H5N1 virus (331) were boosted with a single 90 mg dose of subvirion clade 1 A/Vietnam/1203/04 H5N1 vaccine around eight years later (351). Among the recombinant H5 vaccine recipients (i.e., primed), the response rate was 68%, compared with 23% in naı¨ve subjects (334) suggesting that memory responses to earlier H5 vaccine are preserved, and can be boosted by single vaccination with future virus variant. Thus pre-pandemic priming, followed by a single-dose booster of post pandemicspecific vaccine when it is available, may optimize use of limited vaccine supplies during the first pandemic wave.

VACCINES FOR NOVEL H1N1 VIRUS Human infections with novel H1N1 influenza viruses of swine origin were first identified in April of 2009. The prototype virus of this outbreak, A/California/04/2009 virus, is a so-called quadruple reassortant, deriving the PA and PB2 gene segments from avian influenza A viruses of North American lineage, the PB2 gene segment from a human H3N2 virus, the HA, NP, and NS gene segments from a swine virus of North American lineage, and the NA and M gene segments from a swine virus of Eurasian lineage (352). These H1N1 viruses exhibit antigenic novelty and efficient transmission [demonstrated in ferret animal models (353,354)], necessary attributes for a new pandemic influenza virus. In the *50-day period from their identification on April 24, 2009 until June 11, 2009, global spread of the novel H1N1 viruses reached the criteria for stage 6 of the World Health Organization pandemic stages, and the outbreak was officially declared a pandemic. The H1 HA of the novel H1 virus is an example of a classical swine H1 HA, and is highly divergent from the HA of seasonal H1 influenza A viruses. There is an approximately 25% difference between the HAs of A/California/04/09 and the seasonal H1 of A/Brisbane/08 virus on an amino acid level, with the changes mostly concentrated in known antibody binding epitopes in the globular head of the HA1 component. Antigenically, the A/California/04/09 HA is most closely related to the HA of the swine A/New Jersey/76 virus and to H1 influenza viruses circulating early after the introduction of H1 viruses in humans in 1918. There is little or no cross reactivity between the novel and seasonal H1 HAs using hemagglutination-inhibition (HAI) assays, and unexposed persons less than 60 years of age do not have detectable antibody against the A/California/04/09 virus. Neither seasonal live nor inactivated influenza vaccines induce immune responses that can recognize or would be expected to provide significant protection against the novel H1 virus (355). However, epidemiologic data have suggested that seasonal vaccination might have provided some protection against severe disease during the spring outbreak in Mexico City (356). Observations so far suggest that these viruses cause typical influenza. However, there is a striking difference in the age distribution of cases of novel H1N1 viruses, with the bulk of the disease seen in children and young adults, and relatively little disease in the elderly (357,358). The severity of the disease appears to be roughly similar as well, but because of the extraordinarily high attack rates, the total burden on the health care system has been overwhelming in some localities, and there are many cases requiring hospitalization and intensive care unit care. Pregnancy, obesity, and possibly ethnicity have been identified as potential risk factors for severe disease. Whereas current

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isolates are generally sensitive to neuraminidase inhibitors, there have been rare reports of oseltamivir-resistant viruses. Rapid deployment of effective vaccines against novel H1 viruses will be a critical measure to control the pandemic and mitigate its impact. Currently, specific vaccines against novel H1N1 influenza are generally being licensed by regulatory agencies as a “strain change” based mostly on manufacturing data: Surprisingly, early clinical data indicate that healthy adults between 18 and 64 years of age respond strongly to a single dose of 15 ug of unadjuvanted subvirion inactivated vaccine (359). Similar data from other studies of inactivated vaccines have been reported in press releases, including the announcement by NIAID that 96% of healthy adults and 76% of children from 10 to 18 years of age developed serum HAI antibody titers of 40 or greater within 14 days of receiving a single dose of inactivated vaccine, but that less than 50% of children less than 10 responded to a single dose. Based on the labeling for seasonal vaccine, it is expected that both inactivated and live vaccines will be recommended as a single dose in individuals 9 years of age and above, and as a two dose schedule in children under 9 with no history of previous influenza vaccination. Current recommendations are that the first tier of vaccine be used in pregnant women, individuals 24 years of age and younger, individuals 25 to 64 years with chronic health conditions, and health care workers and emergency services personnel (360).

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342. Stephenson I, Bugarini R, Nicholson KG, et al. Cross-reactivity to highly pathogenic avian influenza H5N1 viruses after vaccination with nonadjuvanted and MF59-adjuvanted influenza A/ Duck/Singapore/97 (H5N3) vaccine: a potential priming strategy. J Infect Dis 2005; 191:1210–1215. 343. Leroux-Roels I, Borkowski A, Vanwolleghem T, et al. Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial. Lancet 2007; 370:580–589. 344. Li S, Liu C, Klimov A, et al. Recombinant influenza A virus vaccines for the pathogenic human A/Hong Kong/97 (H5N1) viruses. J Infect Dis 1999; 179:1132–1138. 345. Chen H, Subbarao K, Swayne D, et al. Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate. Vaccine 2003; 21(17–18): 1974–1979. 346. Stephenson I, Nicholson KG, Gluck R, et al. Safety and antigenicity of whole virus and subunit influenza A/Hong/Kong/1073/ 99 (H9N2) vaccine in healthy adults: phase I randomized trial. Lancet 2003; 362:1959–1966. 347. Atmar RL, Keitel WA, Patel SM, et al. Safety and immunogenicity of nonadjuvanted and MF59-adjuvanted influenza A/H9N2 vaccine preparations. Clin Infect Dis 2006; 43:1135–1142. 348. Rowe T, Abernathy RA, Hu-Primmer J, et al. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol 1999; 37(4):937–943. 349. Stephenson I, Wood JM, Nicholson KG, et al. Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin. J Med Virol 2003; 70(3):391–398. 350. Proposed draft guidance on clinical data needed to support the licensure of pandemic vaccines against influenza. 2007. Accessed October 1, 2007. Available at: http://www.fda.gov/cber/gdlns/ panfluvac.htm. 351. Goji NA, Nolan C, Hill H, et al. Immune respones of healthy subjects to a single dose of intramuscular inactivated influenza A/Vietman/1203/04 (H5N1) vaccine after priming with an antigenic variant. J Infect Dis 2008; 198(5):635–641. 352. Dawood FS, Jain S, Finelli L, et al., Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009; 360:2605–2615. 353. Munster VJ, de Wit E, van den Brand JM, et al. Pathogenesis and transmission of swine-origen 2009 A(H1N1) influenza virus in ferrets. Science 2009; 325:481–483. 354. Maines TR, Jayaraman A, Belser JA, et al. Transmission and pathogenesis of swine-origin 2009 (H1N1) influenza viruses in ferrets and mice. Science 2009; 325:484–487. 355. Hancock K, Veguilla V, Lu X, et al. Cross-reactive antibody responses to the 2009 pandemic H1N1 influenza virus. N Engl J Med 2009; 361. [Epub ahead of print]. 356. Garcia-Garcia L, Valdespino-Gomez JL, Lazcano-Ponce E, et al. Partial protection of seasonal trivalent inactivated vaccine against novel pandemic influenza A/H1N1 2009: case-control study in Mexico City. BMJ 2009; 339:b3928. 357. Chowell G, Bertozzi SM, Colchero MA, et al. Severe respiratory disease concurrent with the circulation of H1N1 influenza. N Engl J Med 2009; 361:674–679. 358. Jain S, Kamimoto L, Bramley AM, et al. Hospitalized Patients with 2009 H1N1 Influenza in the United States, April–June 2009. N Engl J Med 2009. NEJMoa0906695. 359. Greenberg ME, Lai MH, Hartel GF, et al. Response after one dose of monovalent 2009 influenza A (H1N1) vaccine - preliminary report. N Engl J Med 2009; 361. [Epub ahead of print]. 360. CDC. Use of influenza A (H1N1) 2009 monovalent vaccine recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2009; 58:1–8.

52 Chimeric Vaccines Against Japanese Encephalitis, Dengue, and West Nile Konstantin V. Pugachev Sanofi Pasteur, Cambridge, Massachusetts, U.S.A.

Farshad Guirakhoo Sanofi Pasteur, Marcy l’Etoile, France

Dennis W. Trent Xcellerex, Inc., Marlborough, Massachusetts, U.S.A.

Thomas P. Monath Kleiner Perkins Caufield & Byers, Pandemic and Biodefense Fund, Menlo Park, California, U.S.A.

INTRODUCTION Japanese encephalitis (JE), dengue (DEN), and West Nile (WN) viruses are among the most important human pathogens in the Flavivirus genus of the Flaviviridae family. This group of small enveloped RNA viruses includes approximately 70 members, 38 of which have been associated with human illnesses. In addition to the Flavivirus genus, the Flaviviridae family also includes the Pestivirus genus containing several veterinary pathogens that have a worldwide economic impact, such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV), the Hepacivirus genus that includes hepatitis C virus (HCV), an important human pathogen, and several recently identified hepatitis GB viruses not linked to human disease. Although currently grouped within the Flaviviridae family, pestiviruses and hepaciviruses differ significantly from representatives of the Flavivirus genus in terms of their life cycle, genome organization, processing of viral proteins, etc. (1). Therefore, the chimeric vaccine development approaches discussed in this chapter may not be easily applicable to viruses other than those in the Flavivirus genus. This chapter uses the term ‘‘flavivirus’’ to refer to members of the Flavivirus genus only. With a few exceptions, flaviviruses are arthropod-borne viruses (arboviruses) transmitted by mosquitoes and ticks. On the basis of antigenic cross-reactivity and genome sequence similarity, flaviviruses are grouped into four distinct complexes (Table 1): the yellow fever (YF) complex containing YF virus as its sole member; the JE complex [JE, WN, St. Louis encephalitis (SLE), Kunjin (KUN), Murray Valley encephalitis (MVE) viruses, etc.]; the DEN complex that includes the four serotypes of DEN viruses (DEN types 1–4); and the tick-borne encephalitis (TBE) complex [TBE, Kyasanur forest disease (KFD), Langat (LGT), Powassan, louping ill viruses, etc.] (2). There are no antiviral drugs for the treatment of flavivirus infections, although development of new therapeutics has recently accelerated (3), and vector eradication programs have been inefficient in controlling YF, DEN, and the flaviviral encephalitides

(4). Therefore, vaccination of people that live in or travel to endemic areas is the most effective means of protection against these diseases. There are currently no licensed vaccines against DEN and WN encephalitis. The vaccines that are currently available against YF, JE, and TBE, although efficacious, may still benefit from improvements in terms of their safety, efficacy, manufacturing cost, and/or use of acceptable cell substrates by implementation of new molecular biology and cell biology technologies. Several new molecular approaches (recombinant subunit vaccines, DNA vaccines, viruses attenuated using various genetic manipulations) as well as the classical approaches (killed-virus and empirically attenuated vaccine strains) are currently being explored to create DEN, WN, and new JE vaccines (5–8). This chapter will focus on the construction of live chimeric vaccines, particularly those generated using the ChimeriVax1 technology developed by Sanofi Pasteur.

FLAVIVIRUS STRUCTURE AND REPLICATION Flavivirions are spherical particles approximately 50 nm in diameter, the structure of which has been defined in detail with X-ray crystallography and cryoelectron microscopy (1) (Fig. 1A). The genome is a single-stranded RNA molecule of positive polarity of about 11,000 nucleotides (nt) in length. It contains a long open reading frame (ORF) flanked by 50 and 30 untranslated terminal regions (UTRs), approximately 120 and 500 nt in length, respectively. The ORF encodes a polyprotein precursor that is cleaved co- and posttranslationally, resulting in individual viral proteins. The virus proteins are encoded in the order: C-prM/M-E-NS1-NS2A/2B-NS3-NS4A/4B-NS5, where C (core), prM/M (pre-membrane/membrane), and E (envelope) are the structural proteins, that is, the components of viral particles, and NS are the nonstructural proteins functioning in intracellular virus replication (Fig. 1B). In infected cells, the genomic RNA is translated, and processing of the polyprotein begins with translocation of the

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Table 1 Major Human Flavivirus Pathogens and Available Vaccines Available vaccines

Serocomplex Virus

Principal vector

Inactivated

Live attenuated

YF

YF

Mosquito

N/A

þ

JE

JE

Mosquito

þ

þ

WN





St. Louis encephalitis Murray Valley encephalitis Kunjin DEN types 1–4 viruses TBE (Russian spring-summer encephalitis and Central European encephalitis) Kyasanur forest disease

    þ

    

DEN TBE

Mosquito Tick

þ

Comments The highly successful 17D vaccine (produced in 9 countries) Inactivated mouse brain or cell culture derived (Japan, etc.), and S14-14-2 live attenuated (China) No human vaccines; several veterinary vaccines available, including live ChimeriVax1-WN01-based vaccine (Intervet)

Cell culture derived (Austria, Germany, Russia) Cell culture derived (India)

Pathogens causing the most significant morbidity and mortality are in bold. Abbreviations: YF, yellow fever; JE, Japanese encephalitis; DEN, dengue; TBE, tick-borne encephalitis; WN, West Nile.

Figure 1 Flavivirus structure. (A) Organization of the virion. Each viral particle contains a nucleocapsid composed of one genomic RNA molecule and the C protein. Nucleocapsid is surrounded by a lipid membrane with embedded M and E proteins. (B) Organization of the genome. The positive-sense genomic RNA molecule encodes a long open reading frame, which is translated into a polyprotein precursor. Most of the individual viral proteins are produced by cleavages of the polyprotein by cellular signalase (black arrows) and the viral NS2B/NS3 protease (open arrows). The prM protein (precursor for M) is cleaved by a furin-like protease shortly prior to particle release. The C protein generated by the signalase cleavage (Cintracellular) can only be found inside infected cells. Virions contain a crboxy-terminally truncated form of C (Cvirion) that is generated by the viral protease and lacks the signal peptide for prM. Abbreviations: C, capsid; M, membrane; E, envelope; prM, premembrane.

prM sequence into the lumen of endoplasmic reticulum (ER), followed by translocation of E and NS1 sequences. Aminotermini of prM, E, and NS1 are generated by signalase cleavages. The three glycoproteins, prM, E, and NS1, remain carboxy-terminally anchored in the ER membrane. It is

unknown which cytoplasmic protease is responsible for the downstream NS1/NS2A cleavage; however, it is possible that this cleavage occurs autoproteolytically. The N-termini of most other NS proteins are generated by the viral NS2B/NS3 serine protease in the cytoplasm of the infected cells, with the

Chapter 52: Chimeric Flavivirus Vaccines

exception of the N-terminus of NS4B that is formed by a signalase cleavage. The viral protease is also responsible for generating the C-terminus of the mature C protein (Cvirion). Newly synthesized positive strand genomic RNA and the C protein form a nucleocapsid, which acquires an envelope containing the embedded prM and E proteins by budding into the ER lumen. The mature M protein is produced by cleavage of prM shortly prior to virus release by a cellular furin-like protease (9). The role of the glycoprotein NS1 in the biology of flaviviruses was unclear until recently. It appears that NS1 is essential for viral RNA synthesis (10,11), and its secreted form has been speculated to be involved in virus spread in vivo and/or disease pathogenesis and evasion of immune responses. Recent studies have demonstrated that the WN virus NS1 protein interferes with the complement system (12). In addition to the continuing molecular studies on the structure/function of viral proteins and mechanisms of viral RNA replication (1), another active area of flavivirus research is the elucidation of mechanisms of virus-host interactions, such as the role of genetic determinants in resistance of the host to flavivirus infection (13), stimulation or evasion of innate and adaptive immune responses (14), and pathogenesis of flavivirus diseases. For example, several NS proteins have been implicated in inhibiting the induction of interferons (INF) and INF signaling (14–18). The E protein is the main functional protein of the envelope responsible for receptor binding and membrane fusion. This protein plays a dominant role in the induction of neutralizing antibodies that are the principal mediators of protective immunity (2). Virus-specific CD8þ and CD4þ T-cell responses, including cytotoxic T-lyphocyte (CTL) responses, against numerous T-cell epitopes scattered throughout both the structural and NS proteins are also considered essential for protective immunity (19–22).

FLAVIVIRUS DISEASES AND AVAILABLE VACCINES The available information on the mechanisms of pathogenesis of flavivirus diseases has been reviewed (2,3,25,39). After an infectious mosquito or tick bite, virus replication occurs locally in the inoculation skin site and in draining lymph nodes. Virions then disseminate to secondary sites where further replication contributes to viremia and can cause damage to visceral organs (YF, DEN viruses), while encephalitogenic viruses (WN, JE, TBE) can invade the brain, which, in some cases, results in pathology of the CNS. Mechanisms of brain invasion are not well understood, and cell receptors that can mediate different types of flavivirus tropism have not been identified. Virus-specific neutralizing antibodies play a major role in protection from disease via preventing or slowing virus dissemination. In addition, cytolytic antibodies against viral proteins on the surface of infected cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) are presumed to mediate clearance of infection. The relative contribution of T-cell immunity to controlling infection remains a matter of speculation (23). T cells could be essential for limiting virus growth by eliminating virus-infected cells or terminating virus replication by the production of antiviral cytokines.

Japanese Encephalitis Virus JE virus causes a serious neurological disease of children in Asia, with case-fatality rate of 5% to 40%. It is estimated that

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more than three billion people live in regions where JE virus is endemic. In the last 25 years, the incidence has increased in many countries, and JE has extended its geographical range to areas in Asia and northern Australia that were previously free from this disease (24,25). More than 35,000 JE cases are officially reported each year by the World Health Organization (WHO), of which 5000 to 10,000 are fatal. This disease remains a serious threat to unvaccinated travelers to endemic countries. A high proportion of survivors suffer from neurological and psychological sequelae. The virus is transmitted from infected animals, mainly domestic pigs and birds, to humans by Culex mosquitoes. The use of a mouse brain-derived, formalin-inactivated vaccine (Biken, Japan) (Table 1) has significantly reduced disease rates in Japan, Republic of Korea, Taiwan, Sri Lanka, and parts of Thailand and Vietnam. The vaccine is 91% effective and is administered in two primary doses, one booster at one year and subsequent boosters every three years. It has been associated with 0.6% rate of allergic reactions in adults, sometimes severe, and is costly to manufacture. It may soon be replaced by a Vero cell-derived inactivated version under development. Other inactivated JE vaccines produced with virus grown in cell culture have been developed in China and Japan. A live attenuated SA14-14-2 vaccine produced in primary hamster kidney cells has been used in China, with 50 to 60 million doses administered annually (24,25). The highly attenuated SA14-14-2 vaccine has been approved by regulatory agencies in South Korea, Nepal, Sri Lanka, and India. Although multiple doses are generally employed in immunization schemes (e.g., in China), new evidence supports efficacy after a single dose (26,27).

Dengue Virus DEN is a major public health problem of the tropics (2). The incidence and geographic distribution of the disease in tropical and subtropical regions of the world have risen dramatically in the last 40 years (4). Since 1970, the entire tropical world has become hyperendemic for DEN, meaning that all four DEN serotypes co-circulate, while prior to 1970, DEN was only hyperendemic in Asia. This has resulted in frequent and intense epidemics and increased severity of disease. Over two billion people in tropical Asia, Africa, Pacific Islands, Australia, and the Americas are at risk of DEN virus infection. Annually, up to 100 million cases of DEN fever and 450,000 cases of the more severe form of the disease, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) occur. The majority of severe cases occur in young children living in Asia, who suffer a case-fatality rate of approximately 5% (varying locally between 1% and 40% depending on the quality of supportive care). The virus is transmitted to humans by Aedes aegypti (and in some areas Aedes albopictus) mosquitoes. The amino acid homology between the four serotypes is 63% to 68% (and up to 77% in the E protein), compared with 44% to 51% between DEN and other flaviviruses. Infected individuals develop lifelong homotypic immunity, but cross-protection against viruses of the other serotypes is short-lived, lasting less than 12 weeks (28). As a result, people are often infected several times, each time with a different serotype of virus. Such secondary infections with a different DEN virus serotype more frequently result in DHF/DSS. These observations support the theory of immune enhancement of heterologous serotype virus infection due to antibody-dependent enhancement (ADE) of virus replication (29,30) and exacerbation of symptoms by preexisting cellular immunity (20,31). Subneutralizing concentrations of

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antibodies recognizing the conserved fusion peptide of the E protein were recently shown to significantly increase replication of DEN viruses in Fc-receptor bearing cells in vitro and to increase viremia in monkeys, and modifications in the antibody Fc region could abrogate the ADE activity (32). This observation could lead to a new antibody-based therapy of DEN disease. Both virus-specified and host-related factors may influence the severity of the disease, because only approximately 3% of persons with secondary infections develop DHF. Alternatively, DHF may be caused by particularly virulent strains (33,34), and it has been suggested that anti-NS1 antibodies may cross-react with fibrinogen, thrombocytes, and endothelial cells triggering hemorrhage (35). It is generally accepted that a DEN vaccine must be tetravalent, capable of inducing a robust protective immunity against all four serotypes simultaneously, as well as long-term memory and persistence of neutralizing antibodies. Despite more than 60 years of extensive research efforts, no licensed DEN vaccine is yet available, although several promising vaccine candidates, including empirically attenuated DEN viruses, are in preclinical and clinical development (5–8).

West Nile Virus Since the unprecedented introduction of WN virus in 1999 from the Middle East to the New York City area (36), the virus has rapidly spread through North America, the Caribbean, and Mexico, and recently reached continental South America. WN virus is endemic in Africa, the Indian subcontinent, parts of Europe, Southern Russia, Central Asia, and the Middle East. The human disease varies from mild DEN-like illness to fatal meningoencephalitis, with the most severe illness occurring in the elderly (2,3). In the United States, disease incidence peaked in 2003, with 9862 reported cases, approximately one-third of which were accompanied by neurological symptoms, and 264 deaths. In 2004 to 2008 the incidence declined approximately three-fold, with 100 to 177 deaths annually, and it was hoped that the virus was genetically adapting to the new environment and becoming less virulent (37). Surveillance data from the 2007 WN season in the United States recorded another year with substantial disease burden as 3630 cases and 124 deaths were reported (38). As of October 7, 2008, 1030 cases and 24 deaths were reported in the United States for 2008. The emergence of WN in North America has spurred extensive interest in the development of human and veterinary vaccines. While there is still no human vaccine available, in 2006 Intervet Inc. (Millsboro, Delaware, U.S.) received approval of the first live attenuated chimeric single-dose vaccine for horses based on the ChimeriVax-WN01 virus (see below).

Yellow Fever YF virus, the prototype member of the Flavivirus genus, was first clinically recognized in the 17th century and remained one of the most dreaded diseases in tropical Africa and South America until the 20th century when an effective vaccine was developed. The symptoms of this lethal hemorrhagic fever transmitted to humans by Aedes mosquitoes include fever, hepatic, renal, and myocardial injury, hemorrhage, prostration, and shock. Today, because of incomplete vaccination coverage and mosquito reinfestation, YF still affects approximately 200,000 persons a year, and continues to be a threat to travelers (39). Wild-type YF virus, strain Asibi, was first isolated in 1927 by inoculation of a rhesus monkey with blood from a patient in Ghana. In 1937, Theiler and

Smith reported successful attenuation of this virus by multiple passages in mouse and chick embryo tissues that yielded the 17D vaccine strain (40). In the approximately 70 years since its development, the 17D vaccine has been administered to over 400 million people with a remarkable history of safety and efficacy. The 17D vaccine is currently manufactured in several countries (France, United States, Russia, Switzerland, Senegal, Colombia, China, and India) in embryonated chicken eggs under standards established by WHO, and a sub-strain of 17D (called 17DD) is produced in Brazil. The vaccine is well tolerated, with few, usually mild, side effects such as injection site pain, redness, headache, etc. After vaccination, a low viremia is detectable during the first few days, not exceeding 2 log10 pfu/mL. Because of a low viremia in vaccinated individuals and the fact that, in contrast to wild-type virus, the 17D virus does not replicate in mosquitoes, vaccination cannot lead to dissemination of 17D. Vaccination is contraindicated in persons with immune deficiency disorders or those taking immunosuppressive medications. Owing to the vulnerability of infants, the vaccine is not recommended in children younger than nine months. Except when disease is epidemic, pregnancy is generally regarded as another contraindication, as congenital infection has been shown to occur at rate of 1% to 2%, although this has not clearly been associated with any harm to the fetus (39). The period of onset of immunity is short. Ninety percent of vaccinees develop protective levels of YF-neutralizing antibodies by day 10, and 99% by day 30 after vaccination. Immunity appears to be lifelong after a single dose; therefore, the 17D vaccine has been regarded as one of the strongest immunogens ever developed. This is probably due to the fact that 17D virus infects dendritic cells, the main antigenpresenting cells, and stimulates strong polyvalent immune responses through activation of multiple Toll-like receptors (41,42). All the above features of the 17D vaccine validate the use of YF 17D virus in the construction of novel, genetically engineered vaccines against other flavivirus diseases.

FLAVIVIRUS CHIMERAS NOT BASED ON THE YF 17D BACKBONE Dengue Intertypic Chimeras The introduction of methods of reverse genetics, or infectious clone technology, opened a new chapter in RNA virus research. An infectious clone is a DNA copy of a viral RNA genome, which is stably cloned (most frequently in bacteria), and can be easily manipulated in vitro. To initiate virus replication, the cDNA template is converted to RNA by in vitro transcription, and appropriate substrate cells are transfected with the RNA transcripts. Alternatively, cells are directly transfected with appropriately designed plasmid DNA. The first flavivirus infectious clone was reported in 1989, for YF 17D virus (43). Since then, infectious clones have been developed for many disease-causing flaviviruses, with the exception of DEN type 3 (DEN3). Infectious clones of flavivirus genomes are now used as tools to construct genetically engineered vaccines including chimeric flavivirus vaccine candidates. The construction of the first viable flavivirus chimera was reported in 1991 by Bray and Lai of the National Institutes of Health (NIH), who replaced the entire structural region, the C-prM-E genes, in the infectious clone of DEN4 (wild-type strain 814669) with the corresponding C-prM-E cassettes from DEN1 (Western Pacific strain, WP) and DEN2 [New Guinea C (NGC), a laboratory strain neurovirulent for mice] (44). Three-day-old suckling mice inoculated intracerebrally (IC) with both the

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DEN2 NGC and chimeric DEN4/DEN2 viruses developed encephalitis and died. There was, however, a three- to five-day delay in death caused by the chimera, while both the DEN1 WP and DEN4/DEN1 viruses were not neurovirulent. Later, DEN4/ DEN2 chimeras containing the prM-E cassette or only NS1 gene from DEN2 in place of the corresponding genes in the DEN4 backbone were successfully generated, while attempts to produce a chimera containing DEN2 C-prM-E-NS1 genes failed (45). A DEN4/DEN3 chimera containing the C-prM-E genes from a wild-type DEN3 strain CH53489 was also obtained (46). These studies demonstrated the possibility of engineering viable intertypic DEN chimeras and also yielded valuable information on genetic determinants of neurovirulence in mice (47). Subcutaneous (SC) inoculation of rhesus monkeys at a dose of 3  105 pfu of either the DEN4/DEN1C-prM-E or DEN4/ DEN2prM-E chimera induced detectable, short-lived viremias. Immunization resulted in high titers of homologous DEN typespecific neutralizing antibodies (1:640 to 1:1280), which were similar to titers observed in control animals inoculated with the DEN1 and DEN2 parents. Challenge of monkeys immunized with the DEN4/ DEN1C-prM-E or DEN4/DEN2prM-E chimeras at 66 days postimmunization with the corresponding wild-type DEN viruses demonstrated no viremia in the majority of immunized animals, while high titer viremias were observed in all unimmunized controls. Similarly, monkeys immunized with a mixture of the two chimeras (DEN4/DEN1 C-prM-E and DEN4/ DEN2prM-E) developed high titers of both DEN1- and DEN2specific neutralizing antibodies (generally 1:320 to 1:640). The animals were solidly protected from challenge with both wildtype DEN1 and DEN2, even despite the fact that the DEN4/ DEN2 chimera clearly outgrew the DEN4/DEN1 chimera in the doubly immunized animals as evidenced by the analysis of post-immunization viremias (48). These data suggested that developing a tetravalent DEN vaccine composed of chimeric viruses based on the genetic background of one flavivirus is possible. The DEN4 virus used in these studies (which would be also the DEN4 component in a tetravalent vaccine formulation, along with three DEN4-based chimeras) is pathogenic for humans and transmissible by mosquitoes. The DEN4 backbone–based approach to DEN vaccine has been pursued by scientists at the NIH and the Food and Drug Administration (FDA) (49–52). To increase safety in humans and decrease the rate of replication in mosquitoes, an attempt was made to attenuate the DEN4 virus by introducing large deletions in its 30 untranslated region (53). A DEN4 variant with a 30-nt deletion (designated D30) was chosen for evaluation in humans (51), and it was proposed that a tetravalent vaccine could be made on the basis of wild-type DEN strains containing this deletion or intertypic chimeras constructed using the DEN4D30 backbone. Some of the published data suggest that the D30 deletion may not be sufficiently attenuating, and additional modification of the backbone is necessary (50,54). The DEN-4D30 virus was associated with mild adverse reactions in a high percentage of vaccinees, even when the vaccine was given at a low dose (e.g., rash in 75% of recipients of a 10pfu dose), and occasional elevations in blood level of alanine aminotransferase at higher doses suggested replication of the virus in the liver (52). In addition, the D30 mutation did not attenuate DEN3 virus in monkeys, and did not reduce replication of DEN3 and DEN1 in mosquitoes (49,55). New vaccine candidates are being further developed and the possibility of using both chimeric and non-chimeric viruses in tetravalent

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mixtures delivered in one- or two-dose regimens is being explored (56). Attenuated DEN1D30 and a chimeric DEN4D30/DEN2 variant have been tested in DEN-naı¨ve adult volunteers at a dose of 103 pfu. Notably, most vaccinees seroconverted to DEN1 or DEN2, respectively, and maintained significant antibody titers throughout the six-month trial duration demonstrating high durability of immune response (57,58). A highly attenuated DEN4D30/DEN1 candidate is also available and has been tested in rhesus monkeys (59). Another promising chimeric approach to tetravalent DEN vaccine has been developed by scientists at the U.S. Centers for Disease Control and Prevention (CDC). Their chimeras are based on the backbone of the attenuated DEN2 PDK-53 virus, which is more attenuated and safer as a chimeric vaccine vector than the DEN4 backbone described above. The PDK-53 DEN2 virus strain was originally developed at Mahidol University (Bangkok, Thailand) by multiple passages of a wild-type DEN2 virus isolate in primary dog kidney cells. This chimeric method had been facilitated by the finding that all attenuating determinants in DEN2 PDK-53 virus map to the backbone, outside the prM-E genes, which can be replaced with heterologous DEN counterparts. PDK-53-based chimeras have been tested in mice and shown to be highly attenuated and immunogenic (7,60). PDK-53 DEN2/DEN1 vaccine variants against DEN1 induced DEN1-specific neutralizing antibodies in cynomolgus monkeys, without viremia. Most immunized monkeys were protected from wild-type DEN1 virus challenge (as well as DEN2) as judged by the analysis of post-challenge viremia (61).

Chimeras Between Unrelated Flaviviruses The successful construction of intertypic DEN chimeras described above stimulated experimentation to develop chimeras between unrelated flaviviruses from different serocomplexes. The first report of a viable chimera between two genetically distant flaviviruses, a mosquito-borne DEN4 and a TBE virus, was published by Pletnev and coworkers in 1992 (62). The DEN4 backbone described above (from the DEN4 wild-type strain 814669) was used in these experiments. The DEN4/TBE chimera contained the prM-E genes of the Far Eastern strain Sofjin of TBE virus. The chimera grew efficiently in simian LLC-MK2 cells but not in mosquito C6/36 cells, in contrast to the parental DEN4, which grows more efficiently in mosquito cells than in simian cells. Interestingly another chimeric variant, containing the TBE C-prM-E genes, was also viable but did not replicate efficiently compared with the prME chimera. This may be due to a number of problems that include inefficient encapsidation of the hybrid genomic RNA by the TBE-specific C protein, disruption of viral RNA cyclization essential for viral RNA synthesis (63) and inefficient cleavage at the C-terminus of the TBE-specific C protein by the DEN4-specific viral protease that generates the mature form of C (Cvirion) (Fig. 1B) (64–66). Attempts to generate other chimeras containing TBE-specific prM-E-NS1 or E-NS1 cassettes, or singly C, E, or NS1 genes, did not result in viable viruses (62). As discussed above, a DEN4/DEN2NS1 chimera was viable (45). Thus, in contrast to closely related DEN types, NS1 is not easily interchangeable between distant flaviviruses, possibly because NS1 is a component of viral RNA polymerase (11), which could be highly constrained for structural compatibility of all participating proteins. Even though the efficiently replicating DEN4/TBEprM-E chimera contained the envelope of an encephalitogenic TBE

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virus, it was not neuroinvasive when inoculated into mice by the peripheral route (62,67). The virus was immunogenic in mice. Because it remained neurovirulent for mice after IC inoculation, another chimera was constructed that contained the prM-E genes from LGT virus (68). LGT virus is less virulent for humans than other members of the TBE serocomplex, and immunity to LGT is cross-protective against TBE. In contrast to the DEN4/TBE virus, DEN4/LGT replicated well only in mosquito cells and had to be adapted to mammalian cells by multiple passages. Attempts to generate other chimeric variants containing the LGT-specific C-prM-E, NS1-NS2A, NS1-NS2ANS2B-part of NS3, and NS2B-NS3 cassettes failed, again indicating that the prM-E genes are the only easily interchangeable genes. Mouse neurovirulence of this chimera was significantly reduced compared with the parental LGT virus and DEN4/ TBE (68,69). Immunization of mice (69) and monkeys (70) with DEN4/LGT protected the animals from subsequent challenge with highly virulent TBE virus. The chimera did not replicate in non-hematophagous mosquitoes Toxorhynchites splendens, which are highly permissive for DEN viruses (70). The DEN4 backbone has also been used to create a DEN4/WN chimeric vaccine candidate against WN. The chimera was found to be highly attenuated and immunogenic in mice and rhesus monkeys, particularly its variant with the D30 deletion in the 3 0 UTR described above (71,72), and the DEN4D30/WN virus is about to enter human clinical trials (73). This candidate failed to infect geese, suggesting that chimerization of WN with DEN4 resulted in complete attenuation for avian hosts (73). Its replication in several species of mosquitoes, including Culex tarsalis mosquitoes that are able to transmit WN virus, was generally restricted. However, the chimera was as infectious as wild-type WN for A. albopictus mosquitoes, a species that was introduced into the United States in the 1980s (54). Another chimeric WN vaccine candidate was constructed using the DEN2 PDK-53 backbone. This chimera was shown to be attenuated and immunogenic in the murine model. It efficiently protected immunized mice from a high-dose WN virus challenge (74). One important safety aspect with DENbased vaccine candidates will be to ascertain that cellular immune responses against backbone proteins do not prime some vaccinees for DHF/DSS, the severe DEN illness, if they are infected with a DEN virus type different from the type of chimeric vaccine backbone. This will be important for individuals traveling to DEN endemic countries who had been vaccinated, for example, with DEN4/TBE against TBE in Europe or Russia, or with DEN2/WN against WN in the United States.

CHIMERIVAX VACCINES

Central to the ChimeriVax1 technology is the use of the best flavivirus backbone available, that of YF 17D vaccine virus. This backbone is the main prerequisite for safety and efficacy in humans and low-level replication in mosquitoes precluding uncontrolled dissemination in nature, while heterologous envelopes provide robust humoral and cellular immunity against target pathogens. The ChimeriVax vaccines against JE (ChimeriVax-JE), DEN (ChimeriVax-DEN), and WN (ChimeriVax-WN) were developed by Sanofi Pasteur (formerly Acambis Inc., Cambridge, Massachusetts, U.S.) in collaboration with many colleagues from industry, academia and the U.S. government, as well as clinicians, worldwide. ChimeriVax-JE was developed in collaboration with St. Louis University (SLU,

St. Louis, Missouri, U.S.) and Baxter (Deerfield, Illinois, U.S.). ChimeriVax-DEN was developed in collaboration with SLU. Early preclinical studies on ChimeriVax-DEN and ChimeriVaxWN were supported by NIH grants. These vaccine candidates were highly effective in animals and humans. Currently, they are in phases II and III clinical trials, and thus are the most advanced in terms of testing in humans among all chimeric vaccines under development. ChimeriVax viruses infect dendritic cells, as shown for ChimeriVax-DEN chimeras, which is a prerequisite of a robust, long-lasting immunity (75, 76). In fact, ChimeriVax-DEN viruses infect dendritic cells more efficiently than YF 17D, and stimulate their maturation. This results in the induction of immunostimulatory cytokines, which is consistent with clinical observations of safety and immunogenicity (76). Among DEN vaccines under development, the tetravalent ChimeriVax-DEN1-4 vaccine was the first for which protective efficacy was demonstrated in a monkey challenge model against all four DEN types (77), and among WN vaccines, ChimeriVax-WN was the first to enter human clinical trials, with promising results. The expected product profiles include single-dose application, low rates of (mild) adverse events, and rapid-onset, durable immunity that fit the current needs for vaccines for both travelers and main target populations in endemic countries (e.g., children or the elderly). In addition, the ChimeriVax technology has been applied by Intervet Inc. to develop a live, attenuated chimera vaccine against WN virus for use in horses. The single-dose equine vaccine has been shown to protect horses against severe intrathecal challenge with wild-type WN virus (78–80), and is now commercially available. A ChimeriVax-SLE chimera has been genetically engineered and, if necessary, could be further developed as a vaccine against SLE virus that causes sporadic disease outbreaks in South and Central America and southern and central U.S. states (81). It has been distributed along with ChimeriVaxWN chimera by the CDC to State Health Department Laboratories for diagnosis and epidemiological surveillance of WN and SLE. The biological properties of ChimeriVax vaccines in animal models as well as humans are exemplified by those for ChimeriVax-JE in Table 2. The characteristics are generally representative of other ChimeriVax candidate vaccines. It should be noted that new serious adverse events associated with YF 17D vaccination have recently come to light with improved surveillance, such as adverse neurotropic disease in adults (incidence *1.3–2.5/1 million) and adverse viscerotropic disease resembling classical YF (incidence *2.5/1 million, which could be higher in the elderly) (8). Although rare, these adverse reactions indicate that an improvement in the YF 17D vaccine safety may be necessary, for example, using molecular manipulations. Importantly, the results described below show that ChimeriVax vaccines are more attenuated compared with YF 17D. For instance, neurovirulence of ChimeriVax viruses in mice and monkeys is significantly lower compared with YF 17D, and it is unlikely that chimeras will be able to cause YF-like symptoms.

ChimeriVax-JE Early reports on the construction of intertypic DEN chimeras and DEN4/TBE were a prelude to the ChimeriVax technology illustrated in Figure 2. It started with the work of Chambers and coworkers, who succeeded in the construction of first YF 17D chimeras containing the prM-E genes (but not C-prM-E)

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Table 2 The ChimeriVax1 Technology as Illustrated by Safety and Efficacy Profile of the ChimeriVax-JE Vaccine Compared with the YF 17D Vaccine Model

Parameter

ChimeriVax-JE

YF 17D

Mouse

Neurovirulence in adult mice

Avirulent at IC doses of up to 6.0 log10 pfu

Neuro virulence in suckling mice Illness scorea Brain pathology scorea Viremiab [mean titer/duration (days)] Mean neutralizing antibody titers Protection from lethal JE challenge Infectivity by oral feeding Serious adverse events Common mild adverse events

IC LD50 of 4.9 log10 pfu 0 0.29 1.84/3.3 1:640 to 1:1600c Yes Not infectious None Injection site reactions, flu-like symptoms

Seroconversion rates Effect of anti-vector immunity Viremiab (mean titer/duration)

100% to JE None 1.43/1-2

Mean neutralizing antibody titers in naı¨ve subjects

PRNT50 1:254 (LNI 1.55) and PRNT50 1:128 (LNI 1.38) in the high- and low-dose groups, respectively PRNT50 1:327 (LNI 2.23) and PRNT50 1:270 (LNI 1.62) in the high- and low-dose groups, respectively To be determined in field clinical trials (against JE)

Virulent; IC LD50 of 1.67 log10 pfu IC LD50 of 0.4 log10 pfu 1 1.17 1.93/2.7 1:3225d N/A Not infectious None Injection site reactions, flu-like symptoms 100% to YF N/A 1.6/1-2 (in naive subjects) LNI 3.98

Monkey

Mosquito Humanse

Mean neutralizing antibody titers in YF-immune subjects Protection

LNI 4.05

Provides efficient protection against YF

a

Mean values after IC inoculation; scored as described in Refs. 86 and 87. Values after SC inoculation; mean viremia in log10 pfu/mL/duration in days. c Mean JE-specific titers in groups of animals immunized with graded doses of ChimeriVax-JE ranging from 2.0 to 5.3 log10 pfu/dose. d Mean YF-specific titer in animals immunized with 5.0 log10 pfu of 17D (as described in Ref. 99). e Six YF-immune and six naı¨ve adults received a single SC inoculation of ChimeriVax-JE (5 log10 pfu in the high-dose group, or 4 log10 pfu in the lowdose group) or YF 17D vaccine (5 log10 pfu). Neutralizing antibody titers were measured by the standard PRNT50 test, or the LNI test representing the log10 difference in virus titer between the serum-virus mixtures prepared using post- and pre-vaccination sera (as described in Ref. 89). Abbreviations: JE, Japanese encephalitis; YF, yellow fever; IC, intracerebral; SC, subcutaneous; LD50, 50% lethal dose; PRNT, plaque reduction neutralization test; LNI, log neutralization index. Source: From Refs. 83, 86, 87, 89, 92, 99. b

Figure 2 The ChimeriVax1 technology. The prM-E genes of the YF 17D virus are replaced with their analogs from a heterologous flavivirus. The C/prM junction is at the signalase cleavage site (Fig. 1B). Chimeric virus is produced following transfection of appropriate culture cells. The heterologous E proteins induce a robust protective immunity in immunized animals against respective flavivirus pathogens. Abbreviations: C, core; YF, yellow fever; E, envelope; prM, premembrane.

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from a wild-type JE virus (Nakayama) or the SA14-14-2 vaccine strain (82). The YF 17D/SA14-14-2 chimera was avirulent in young mice by both IC and intraperitoneal (IP) routes at all tested doses up to 6 log10 pfu, and significantly less neurovirulent in suckling mice compared with YF 17D virus that is lethal for mice of all ages inoculated by the IC route (Table 2). The chimera induced solid immunological protection against challenge with a highly virulent strain of JE virus (83). Importantly, protection in mice was achieved against wild-type viruses belonging to all four major JE virus genotypes (84). The YF 17D/SA14-14-2 chimera was selected as a primary vaccine candidate designated ChimeriVax-JE. This virus grew to high titers, in excess of 7 log10 pfu/mL, in Vero cells acceptable as a substrate for human vaccine manufacture. It also replicated efficiently in tested simian, human, mouse, and mosquito cells. The virus had the antigenic specificity of JE virus, that is, was neutralized by JE-specific antibodies but not by YF-specific antibodies. It was found to be highly stable genetically and phenotypically. Mouse neurovirulence of the virus did not increase following 18 passages in cell culture (MOI 0.1 pfu/cell) or six mouse brain-to-brain passages (83). In a separate study, it was demonstrated that multiple simultaneous reversions to the JE Nakayama sequence in distinct clusters of the E protein were required for reversion of ChimeriVax-JE to a higher neurovirulence in mice (85). Yet, in contrast to JE Nakayama virus, YF 17D/Nakayama chimera was not neuroinvasive, and its mouse neurovirulence profile was similar to YF 17D (82). Extensive testing of ChimeriVax-JE in rhesus monkeys demonstrated that the vaccine virus was highly attenuated for this primate species and less pathogenic than the YF 17D vaccine in all standard tests (86,87). The virus induced a low, self-limited viremia following both IC and SC inoculation, similar to viremias induced by 17D. This is an important feature that minimizes the possibility of neuroinvasion and encephalitis in vaccinees, and reduces the chances of virus spread in nature by feeding mosquitoes. Immunization with doses of ChimeriVax-JE vaccine, as low as 2 log10 pfu and up to 5.3 log10 pfu, elicited high titers of JE-specific neutralizing antibodies of 1:640 to 1:1600 after a single SC inoculation. Animals that received the vaccine were protected from a severe IC challenge with a highly virulent wild-type JE virus strain (86,87) (Table 2). In addition to the standard SC inoculation, the vaccine can be delivered to the epidermis, by skin microabrasion, eliminating the need for needles (88). ChimeriVax-JE has been shown to be well tolerated and highly immunogenic for humans vaccinated with the virus in three phases I and II clinical trials. In one trial, administration of a single dose of 4 or 5 log10 pfu of the virus to YF-immune and naı¨ve volunteers caused no serious adverse events (89). The rates of mild, transient injection site reactions and flu-like symptoms were similar to control groups of subjects that received the YF 17D vaccine. Subjects inoculated with the chimera in both dose groups developed a transient, low-titer viremia similar in magnitude and duration to that following 17D immunization. The rates of seroconversion to JE were 100% in both high- and lowdose groups in both naı¨ve and YF-immune subjects. The mean JE-specific neutralizing antibody titers were higher in the highdose groups (1:254 and 1:327 in naı¨ve and YF-immune subjects, respectively) than in the low-dose groups (1:128 and 1:270 in naı¨ve and YF-immune subjects, respectively), and also higher in YF-immune than naı¨ve individuals (Table 2). These data dispel

the concern that YF 17D anti-vector immunity could limit the usefulness of ChimeriVax vaccines in regions where the general population were either immunized against YF or were infection immune. Anti-vector immunity in the case of ChimeriVax viruses could involve cytotoxic T-lymphocyte responses to YF 17D virus NS proteins or cytolytic antibodies against NS1. For comparison, vaccinia recombinants expressing JE virus immunogens failed to induce JE neutralizing responses in vaccinia-immunized subjects (90). In another clinical trial, 10 subjects vaccinated with ChimeriVax-JE were challenged with one standard dose of formalin-inactivated JE vaccine (JE-VAX) as a surrogate for exposure to live virus. The vaccinees demonstrated a significant rise in JE virus-specific neutralizing antibodies (100-fold on day 14 post-challenge), while the control naı¨ve participants showed no or barely detectable antibody levels. Thus, a strong anamnestic immune response, an important prerequisite of vaccine effectiveness, was observed in the vaccinated individuals. In a third study, the ChimeriVax-JE vaccine was equally effective in subjects immunized with five different graded doses ranging from 1.8 to 5.8 log10 pfu (n ¼ 11 to 44 per group), and sera from vaccinees efficiently neutralized Japanese, Chinese, and Vietnamese wild-type strains of JE virus (91). Collectively, these data from clinical trials demonstrate an excellent safety and efficacy profile for ChimeriVax-JE in humans. Phase III safety and efficacy clinical trials have been completed showing an acceptable safety profile and non-inferior immune response of one dose of ChimeriVax-JE to three doses of inactivated mouse brain JE vaccine, and a pediatric study in children in India is underway. A license application for this vaccine is in preparation. An important feature of a successful vaccine is that it is safe for the environment. Similar to the YF 17D virus, and in contrast to the SA14-14-2 parent vaccine virus, ChimeriVax-JE has been shown to be unable to infect Aedes and Culex mosquitoes by oral feeding (92,93). This observation, together with low, short-lived post-inoculation viremia in humans and animals, which is insufficient for infecting feeding mosquitoes, virtually eliminates the possibility of uncontrolled dissemination of the vaccine virus in nature. In contrast, oral polio vaccine viruses, for example, readily spread and recombine with natural polioviruses. In terms of the theoretical possibility of recombination of ChimeriVax vaccines with endemic flaviviruses, it is further minimized by the fact that there are no known, confirmed examples of recombination between flaviviruses in nature, even among the genetically close DEN virus types (94,95). The YF 17D vaccine has been widely in use for 70 years, and there has been no evidence of its uncontrolled spread or recombination with any wild-type flavivirus, including YF. To demonstrate experimentally that recombination in nature between a wild-type flavivirus and a ChimeriVax vaccine virus would result in a recombinant with little potential to cause disease or even survive in nature, artificial recombinants between ChimeriVax-JE and an Australian virus KUN were constructed. The resulting chimeras proved highly attenuated in comparison with the KUN parent. They replicated very poorly in mice and hamsters, were not neuroinvasive, and their neurovirulence in mice was similar to YF 17D and significantly lower than KUN (96). These results further strengthened the point that any recombinants, should they ever emerge, would have little chance to cause disease or spread by successfully competing in nature with wild-type flaviviruses.

Chapter 52: Chimeric Flavivirus Vaccines

ChimeriVax-DEN The first viable YF 17D/DEN chimera (YF 17D/DEN2, subsequently designated ChimeriVax-DEN2) was constructed by Chambers’ group, by insertion of the prM-E genes from a wild-type strain of DEN 2 PUO-218 isolated from a patient in Thailand, and shown to be highly attenuated and immunogenic in mice and rhesus monkeys at Sanofi Pasteur (97). Interestingly, initial attempts to produce a similar chimera by Caufour et al. failed (98). Close examination of the two cloning strategies revealed that Caufour et al. initially followed the approach used in the construction of DEN4/TBE chimera (62). Specifically, they attempted to fuse the 50 end of the DEN2 prM gene with the 30 end of the YF C gene at the Cvirion/Cintracellular viral protease cleavage site and thus the transmembrane signal peptide for prM was DEN2-specific, whereas ChimeriVaxDEN2 virus was engineered to contain the YF-specific signal sequence (Fig. 1B). Because YF and TBE viruses both contain a 20 amino acid-long signal while DEN2 and DEN4 viruses have a shorter 14-amino acid signal, these results indicated that the length of the signal peptide is important for chimera viability. It appears that in DEN4-based chimeras, the short DEN4-specific signal for prM can be replaced with a longer one from another flavivirus, resulting in a viable chimera [although specific amino acid residues in the vicinity of the viral protease and signalase cleavage sites flanking the signal peptide play a role also, as shown for DEN4/WN (71)], whereas the long YF-specific signal needs to be retained in YF 17D-based chimeras. Consistent with this view, when the long YF-specific signal was replaced with the short DEN2-specific signal in ChimeriVax-DEN2, viability was lost (Miller and Arroyo, Sanofi Pasteur unpublished data), and Caufour et al. obtained a viable YF 17D/ DEN2 construct using the YF-specific signal (98). The likely explanation for these observations is that the source (length) of the prM signal peptide in these flavivirus chimeras affects the coordinated fashion of cleavages at the flanking viral protease and signalase sites that is known to be critical for flavivirus replication (64–66). In this regard, it is interesting that a long WN-specific signal was found to be required for viability of PDK-53 DEN2/WN chimera (74). Three other YF 17D/DEN virus chimeras, ChimeriVaxDEN1, ChimeriVax-DEN3, and ChimeriVax-DEN4, were constructed at Sanofi Pasteur (77,99–101). They contain the prM-E genes from wild-type DEN1 PUO 359 (Thailand, 1980), DEN3 PaH881/88 (Thailand, 1988), DEN4 1228 (Indonesia, 1978) strains, respectively. Similar to ChimeriVax-JE, the ChimeriVax-DEN chimeras are avirulent for young mice, and significantly less neurovirulent in suckling mice than the YF 17D virus. These chimeric viruses grow to titers of approximately 107 pfu/mL in Vero cells used for GMP manufacturing. The viruses are highly genetically and phenotypically stable, as very few mutations accumulated during serial passage in cell culture, and there was no increase in mouse neurovirulence after 13 to 18 passages in Vero cells. In rhesus monkeys, the four DEN chimeras administered by the SC route as mono- or tetravalent formulations produced low, brief viremias with peak titers of approximately 2 log10 pfu/mL. In comparison, viremias of the parental wild-type DEN viruses were as high as 4.9 log10 pfu/mL. Strong neutralizing antibody responses of the expected type specificities were induced in sera of immunized animals (99–101). Graded doses of the DEN2 chimera ranging from 2 to 5 log10 pfu/dose were tested and resulted in similar levels of DEN2-neutralizing antibody titers of approximately

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1:320 on day 30, illustrating high immunogenicity of these viruses. Subsequent SC challenge with 5.0 log10 pfu of a wildtype DEN2 resulted in no detectable viremia of challenging virus in any of the immunized animals (97). Testing of tetravalent mixtures of ChimeriVax-DEN1-4 in rhesus monkeys indicated that an appropriate formulation of the components must be determined to achieve uniform antibody responses against all four serotypes. When monkeys were given a mixture of 4.7 log10 pfu of each chimera (99), the ChimeriVax-DEN2 virus induced a higher viremia than the other three. Whereas monkeys seroconverted to all four serotypes, the anti-DEN2 neutralizing antibody titers were higher (1:142, 1:905, 1:127, and 1:71 against DEN1–4, respectively, on day 180). Unequal rates of virus replication upon simultaneous inoculation have been observed previously with other DEN vaccine candidates in both monkeys and human volunteers (48,102). A second inoculation with the same ChimeriVaxDEN1-4 tetravalent formulation resulted in no detectable viremia of any virus, indicating that primary immunization was protective, and the antibody titers became more uniform (1:640, 1:1810, 1:452, and 1:359, respectively). Thus, a more uniform immunity to all four serotypes could be attained by a two-dose vaccination. Importantly, high titers of DEN-specific neutralizing antibodies were induced in both YF-immune and naı¨ve monkeys to confirm that anti-vector immunity is not a concern (99,100). In another experiment, the amount of ChimeriVaxDEN2 was reduced to 3 log10 pfu, to reduce dominance of this candidate, while doses of the other three viruses were 5 log10 pfu each (100). The dose adjustment resulted in a more balanced immune response to DEN1, 2, and 3, but somewhat higher against DEN4 (mean titers of 1:360, 1:400, 1:250, and 1:1400, respectively). Thus, the immune response to the tetravalent vaccine can be regulated by adjusting proportions of its components. Administration of a second tetravalent dose two months after the primary immunization increased antibody titers to all four serotypes. Antibodies in sera of the immunized animals efficiently neutralized various wild-type DEN strains from different geographic regions (100). Plaque-purified cGMP vaccine lots of ChimeriVax-DEN1-4 vaccine candidates for human clinical trials were manufactured at Acambis and Sanofi Pasteur, and tetravalent mixtures were examined for neurovirulence and protective efficacy in cynomolgus monkeys (77). Brain lesions produced by a 5,5,5,5 (log10 pfu for each of the four components) tetravalent formulation after IC inoculation were significantly less severe than those observed with YF 17D (YF-VAX), and there were no nonneural tissue abnormalities. The immunogenicity and protective efficacy of four different tetravalent formulations (5,5,5,5, 5,5,5,3, 3,5,5,3, and 3,3,3,3 log10 pfu of each of the four respective serotypes) were evaluated after a single-dose SC vaccination. Most of the monkeys in the groups, and all monkeys that received equaldose mixtures (5,5,5,5 and 3,3,3,3) seroconverted against all four DEN virus serotypes. All monkeys were protected from challenge with wild-type DEN1-4 viruses at six months post-vaccination, as evidenced by lack of post-challenge viremia, with the exception of one animal from the 5,5,5,3 and another animal from the 3,5,5,3 groups challenged with DEN1 and DEN4 viruses, respectively. Thus, the 5,5,5,5 and 3,3,3,3 formulations of these plaque-purified vaccine viruses were 100% protective. These results demonstrated safety of a recombinant tetravalent DEN vaccine in a formal neurovirulence test and its complete protective efficacy in a monkey challenge model.

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The construction of similar YF 17D/DEN2 and YF 17D/ DEN1 chimeras has been reported by two other groups (98,103–105). The induction of a robust CD8þ T-cell response against the DEN2-specific prM-E envelope proteins was observed in mice (103), and Galler’s group has demonstrated good safety and immunogenicity of their chimeras in monkeys, tested monovalently (104,105). As was the case for ChimeriVax-JE, there is very little potential for transmission of ChimeriVax-DEN vaccine viruses by mosquitoes. A. aegypti, the principal DEN and YF virus vector, and A. albopictus mosquitoes were fed on artificial blood meals containing each of the viruses or a mixture of all four viruses. In contrast to wild-type DEN, the vaccine viruses were invariably highly attenuated with respect to their ability to infect mosquitoes and particularly with respect to dissemination from the gut to the salivary glands (106–108). An initial phase I clinical trial of the ChimeriVax-DEN2 vaccine candidate was conducted to evaluate its safety, tolerability, and immunogenicity in healthy adults with and without prior YF vaccination (n ¼ 14 per study group) (109). The vaccine was well tolerated. Most mild adverse events in the high (5 log10 pfu) and low (3 log10 pfu) dose groups were similar to a control YF-VAX group (5 log10 pfu of YF 17D), and there were no serious side effects. Mean peak viremias in all groups were below 2 log10 pfu/mL. 100% and 92.3% of subjects in the high and low ChimeriVax-DEN2 groups, respectively, seroconverted to DEN2, and 92% of subjects inoculated with YF-VAX seroconverted to YF 17D virus. High serum titers of DEN2 neutralizing antibodies were induced by day 31 (*1:350) and remained similarly high at 6 and 12 months post-immunization with ChimeriVax-DEN2, demonstrating excellent durability of the immune response. Pre-immunity to YF did not interfere with ChimeriVax-DEN2 immunization. T-cell responses against inactivated ChimeriVax-DEN2 antigen were detected using IFNg ELISA in the majority of ChimeriVax-DEN2 immunized subjects. Interestingly, YF-immune subjects inoculated with ChimeriVax-DEN2 (but not YF naı¨ve subjects) seroconverted to all four DEN serotypes. Although the underlying immunological mechanism of this phenomenon needs to be further investigated, this finding may have important practical implications for the development of tetravalent DEN vaccine. Several tetravalent formulations are currently being tested by Sanofi Pasteur in phase I/II clinical trials, and results will be available shortly.

ChimeriVax-WN ChimeriVax-WN was constructed using the prM-E genes from the New York-1999 WN strain. The chimera, referred to as ChimeriVax-WN01, was recovered following transfection of Vero cells and replicated to titers in excess of 7 log10 pfu/mL. This chimera was found to be significantly attenuated for mice when compared with both its WN parent and YF 17D. It was not neuroinvasive, but retained a degree of residual neurovirulence (110). To obtain a more attenuated vaccine candidate for human use, three attenuating SA14-14-2-specific amino acid changes were introduced into the E protein of ChimeriVaxWN01 at residues 107, 316, and 440, resulting in ChimeriVaxWN02 variant. The latter was completely avirulent in adult mice, and dramatically less neurovirulent than YF 17D in suckling mice (p < 0.0001) (110). Neuropathological scores after IC inoculation of both rhesus and cynomolgus monkeys were lower for ChimeriVax-WN02 than YF 17D virus. There

were no abnormalities in hematology and clinical chemistry, and no histological changes were observed in any examined peripheral organ of cynomolgus monkeys following both IC and SC inoculation. Post-inoculation viremia was lower compared with YF 17D in rhesus monkeys, but higher in cynomolgus monkeys, yet within the WHO specifications established for YF 17D vaccine (110,111). The latter observation was associated with a more pronounced early replication of ChimeriVax-WN02 in the skin inoculation site and lymph nodes. Generally, the biodistribution in monkeys of both ChimeriVax-WN02 and YF 17D viruses was similar, as demonstrated using sensitive quantitative PCR. Prominent sites of replication were skin and lymph tissues (as well as the spleen for YF 17D), generally sparing vital organs including the brain (111). The chimera was highly immunogenic and protected immunized monkeys from lethal IC challenge with a high dose (5 log10 pfu) of WN NY99 virus (110). Immunized hamsters were also protected (112). In the first phase I clinical trial in healthy adults, the incidence of adverse events in subjects receiving the ChimeriVax-WN02 vaccine (5 log10 pfu, n ¼ 30; and 3 log10 pfu, n ¼ 15) was similar to the placebo group. Transient viremia was detected in most subjects. All vaccinees developed neutralizing antibodies to WN, and the majority developed WN-specific T-cell responses. Neutralizing antibody response peaked on day 21 at mean titer of approximately 6000 in the 5 log10 dose group, then dropped to approximately 1280 by day 28, and remained stable at this very high level until day 365, which was the last day of the study (111). Phase II safety/immunogenicity studies, including in the elderly representing the main target population, are currently underway. Either Culex and Aedes mosquitoes, including species transmitting WN in the United States, could not be infected by the chimera, or the virus failed to spread to head tissue (113). The virus also failed to infect chickens and fish crows (114). Thus the chimera is highly unlikely to enter a natural transmission cycle with mosquito vectors and birds as amplifying hosts.

CONCLUSION Recent advances of molecular biology have opened doors to the development of new recombinant live flavivirus vaccines. These are currently championed in terms of their high safety and efficacy demonstrated in both animal models and humans by the ChimeriVax vaccine candidates that are based on the most effective and safe flavivirus backbone, that of YF 17D vaccine virus. Within the next few years, it is anticipated that some of the described vaccines, for example, the more clinically advanced ChimeriVax-JE, will be licensed products in use as public health tools preventing human disease and saving lives.

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94. 95. 96.

97.

98.

99.

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53 Vaccines Against Rotavirus Gastroenteritis Richard L. Ward Division of Infectious Diseases, Children’s Hospital Medical Center, Cincinnati, Ohio, U.S.A.

H. Fred Clark and Paul A. Offit Department of Pediatrics, Division of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

Umesh D. Parashar National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, U.S.A.

Roger I. Glass Fogarty International Center, National Institutes of Health, Bethesda, Maryland, U.S.A.

WORLDWIDE IMPORTANCE OF AN EFFECTIVE ROTAVIRUS VACCINE Diarrheal diseases are among the most common illnesses of mankind and the first or second most common cause of death, hospitalization, and doctor visits among children worldwide (1,2). They almost always result from infection, and over the past three decades, more than 25 distinct infectious agents have been identified as etiologic agents of diarrheal diseases. For most children, episodes of diarrhea are self-limiting, the disease is mild, and recovery occurs within several days. However, in some children, the disease can be severe, progressively leading to dehydration, hospitalization, and death. Of the many diarrheal illnesses, rotavirus has been identified as the most common cause of severe diarrhea in children. Several key features define the epidemiology of rotavirus disease and suggest that prevention and control will likely best be achieved with vaccines (3). First, infections are universal and all children worldwide are infected within their first three years of life. This suggests that improvements in food and water sanitation are unlikely to alter the incidence of disease; therefore, other approaches are necessary. Second, initial infections with rotavirus that occur several months after birth are often associated with severe diarrhea. Immunity to disease develops after each rotavirus infection, so second and subsequent infections are usually not associated with illness, and the incidence of rotavirus disease diminishes with increasing age (4). Finally, despite global efforts to diminish the severity of diarrhea through the use of oral rehydration, diarrhea related to rotavirus remains a major cause of hospitalization and death. Global interest in the development of rotavirus vaccines has been driven by the burden of fatal disease in developing countries (Fig. 1) and by medical and societal costs in industrialized countries (5,6). In developing countries, rotavirus is estimated to cause approximately 530,000 (475,000–570,000) childhood deaths each year (Fig. 2). This represents about 5%

of the 10,000,000 deaths worldwide annually among children less than five years of age, or about one death per 270 children born worldwide (ca 135,000,000 births/year) (7). In both developed and industrialized countries, rotavirus accounts for between 20% and 60% of hospitalizations for diarrhea, and recent surveys suggest that one child in every 30 to 120 will be hospitalized for rotavirus diarrhea before his/her fifth birthday. In the United States, efforts to include a rotavirus vaccine in the national immunization schedule were prompted by estimates that rotavirus diarrhea results in 600,000 doctor or emergency room visits, 55,000 to 70,000 hospitalizations, and 20 to 40 deaths each year (6). The cost of this illness has been estimated to exceed US$400 million in medical expenses and more than US$1 billion when societal costs (e.g., parents’ lost work time) are included.

ROTAVIRUS STRUCTURE AND REPLICATION The rotavirus particle (Fig. 3) is ca 100 nm in diameter and has a capsid composed of three concentric protein layers (8,9). The outer layer contains 780 molecules of VP7 and 60 trimers of VP4 (10–12). Both are neutralization proteins and define the G and P serotypes (genotypes) of the virus, respectively. VP4 forms spike-like projections that extend through and 11 to 12 nm beyond the VP7 layer (8,9,13,14). VP4 is anchored to an intermediate layer composed of 780 molecules of VP6 protein (10,11). The innermost layer contains 120 molecules of VP2 protein that interact with 12 molecules each of the viral transcriptase (VP1) and guanylyltransferase (VP3) along with 11 segments of the double-stranded RNA genome. These segments encode the six structural proteins of the virus designated VP1-VP6 and six nonstructural proteins designated NSP1-NSP6 (Table 1). The replication cycle of rotavirus is activated by cleavage of VP4 by trypsin-like proteases but the VP5* and VP8*

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Figure 1 Estimated global distribution of annual deaths due to rotavirus diarrhea in children under five years of age. Source: From Ref. 7.

Figure 3 Computer-generated image of the triple-layered rotavirus particle. The cut-away diagram shows the outer capsid composed of VP4 spikes and VP7 layer, intermediate VP6 layer, and inner VP2 layer surrounding the core containing 11 double-stranded RNA segments and VP1 and VP3 proteins. Source: Courtesy of Dr B.V.V. Prasad, Baylor College of Medicine, Houston, Texas.

Figure 2 Estimated annual global burden of rotavirus infections, episodes of serious disease, and deaths.

products remain virus-associated (15). After attachment to receptors via VP8*, the virion passes into the cytoplasm where the outer capsid proteins are removed (16). This stimulates VP1 to synthesize 11 viral mRNAs that are capped by VP3, extruded from the virus cores, and translated into viral proteins (17). Once viral proteins accumulate, large inclusions or viroplasms are formed in the cytoplasm where assembly of virions is initiated between viral plus strands and several structural and nonstructural proteins (18). Precursor particles formed within the viroplasm evolve into double-layered viral particles with the sequential addition of VP2 and VP6, and subsequently single-stranded RNAs are converted into doublestranded genome segments (19). The double-layered particles then bud into the rough endoplasmic reticulum where final assembly occurs with the addition of VP4 and VP7. Rotavirus replication occurs primarily in the mature enterocytes at the tips of the intestinal villi (20). Following infection of calves and piglets, the cells at the villus tips become denuded (Fig. 4), which results in stunting of the villi (21). Pathology in humans is also associated with damage to intestinal villi. However, non-gastrointestinal rotavirus-associated diseases have been sporadically reported, and several recent publications on viremia suggest the virus has the potential to

spread extra-intestinally (22–25). Therefore, the possibility exists that rotaviruses also cause non-intestinal diseases. However, productive replication of rotaviruses at sites distant from the intestinal mucosal surface has never been conclusively demonstrated.

NATURAL ROTAVIRUS INFECTIONS PROTECT AGAINST SUBSEQUENT ROTAVIRUS DISEASE Rotavirus infection of animals or humans has been shown to be protective against subsequent rotavirus illnesses. In an early study, it was found that human neonates asymptomatically infected with rotavirus were subsequently protected against severe rotavirus disease but were not protected against rotavirus reinfection (26). More recent studies on neonatal rotavirus infection conducted in India provided similar conclusions (27,28). Natural rotavirus infection of infants and young children has been reported in multiple studies to provide at least partial, and sometimes complete, protection against subsequent rotavirus illness. For example, in a cohort study conducted in Mexico, it was found that subjects can become reinfected several times during the first two years of life (4). However, the severity of illness plummeted as infection number increased, and even a single infection reduced subsequent severe rotavirus illnesses by 87%. This occurred even though several rotavirus serotypes circulated concurrently during the evaluation period. The interpretation of these and other observations has led to divergent approaches in rotavirus vaccine development.

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Table 1 Sizes of Rotavirus Gene Segments and Properties of Encoded Proteins Number of base pairs

Encoded protein

Molecular weight of protein (
104)

1

3300

VP1

12.5

2

2700

VP2

10.2

3

2600

VP3

9.8

4

2360

VP4

8.7

5

1600

NSP1

5.9

6

1360

VP6

4.5

7

1100

NSP3

3.5

8

1060

NSP2

3.7

9

1060

VP7

3.7

10

750

NSP4

2.0

11

660

NSP5

2.2

NSP6

1.2

RNA segment

DEVELOPMENT OF ATTENUTATED ROTAVIRUSES AS VACCINE CANDIDATES Animal Rotaviruses as Vaccines The first candidate vaccines evaluated in young children were animal rotavirus strains, an approach still being used today. This approach is based on the finding that transmission of rotavirus disease from animals to humans is relatively uncommon. The approach has met with considerable success since these vaccines have caused few if any symptoms typically associated with rotavirus disease. The first trials were performed using RIT 4237, a serotype G6P[1] bovine rotavirus. This vaccine was safe in Finland, and provided protective efficacy of more than 80% against severe rotavirus disease due to heterotypic human rotaviruses (29). However, studies on this vaccine were terminated when it failed to provide significant efficacy in developing nations (30–32). WC3, a G6P[5] bovine strain, was tested as a vaccine candidate a short time later. After initial promising results (33), this candidate also showed no significant protection in subsequent trials (34,35). In an attempt to make WC3 more serotypically related to human strains, genes encoding the VP4 and VP7 neutralization proteins from human rotaviruses were introduced into WC3 by gene reassortment. This resulted in the development of the pentavalent RotaTeqTM vaccine, one of two rotavirus vaccines being widely licensed in the world today. The simian rotavirus strain RRV developed during this same period also elicited inconsistent protection as a vaccine

Properties of proteins Inner core protein RNA binding RNA transcriptase Inner capsid protein RNA binding Inner core protein Guanylyltransferase Methyltransferase Outer capsid protein Hemagglutinin Neutralization protein Receptor binding Fusogenic protein Nonstructural protein RNA binding IRF regulatory protein Intermediate capsid Group and subgroup antigen Nonstructural protein RNA binding Translational control Nonstructural protein RNA and NSP5 binding Virosome formation Outer capsid glycoprotein Neutralization protein Nonstructural glycoprotein Transmembrane protein Enterotoxin Nonstructural protein Phosphorylated NSP2 and NSP6 binding Nonstructural protein NSP5 binding

(36–39). The G serotype of the G3P[3] RRV strain is shared with human rotaviruses, which can be the dominant circulating strains, and protection was claimed to be more effective when this was the case. As a result, RRV was also reassorted with human strains to incorporate VP7 genes of the other dominant human G serotypes (G1, G2, and G4), thus creating the tetravalent RotashieldTM vaccine. This vaccine was licensed and incorporated into the U.S. infant immunization program in 1998 but was withdrawn by its manufacturer in 1999 after the discovery of a small but significant increase in intussusception (IS) in vaccinees during post-licensure surveillance (40). The only other vaccine candidate developed directly from an animal strain and tested in humans was the G10P[12] lamb strain. This virus was administered to more than 1000 children (aged 6–24 months) in a phase II trial with no evidence of side effects (41). Although no controlled efficacy trials have been reported, this vaccine is presently licensed and sold in China.

Rotaviruses Derived from Humans as Vaccines Several candidate rotavirus vaccines have been obtained from infected humans and most are from neonates. When neonatal rotavirus infections are endemic in some hospitals, they normally cause no disease and, thus, may be naturally attenuated. Furthermore, they have been found to elicit at least partial protection against rotavirus disease. The first of these candidates (M37)

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Human rotaviruses derived from community strains have also been developed as vaccine candidates. Although initial trials with several of these strains suggested that they might be insufficiently attenuated, interest was revived when a circulating strain produced 100% protection after an initial infection (45). Although the 89-12 vaccine derived from one of these circulating viruses was not fully attenuated, even after 33 cell culture passages, it was highly immunogenic and protective in infants (46). After selection of a single virus from within the passage 33 preparation by endpoint dilution followed by several additional cell culture passages of the selected virus, the vaccine remained immunogenic and was reported to be fully attenuated (47,48). This vaccine, called RotarixTM, is the other rotavirus vaccine being licensed worldwide today. A listing of the live rotavirus vaccine candidates that have been evaluated in clinical trials and their current status is presented in Table 2.

THEORIES REGARDING THE EFFECTORS OF PROTECTION AFTER LIVE ROTAVIRUS INFECTION

Figure 4 Normal histologic appearance of ileum from an eight-dayold gnotobiotic piglet. Normal mature vacuolate absorptive cells cover the villi (top). Ileum from an eight-day-old gnotobiotic piglet after oral inoculation with virulent human rotavirus strain Wa (bottom). Severe atropy and early crypt hyperplasia are evident. Source: Courtesy of Dr L.A. Ward, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio.

stimulated poor immune responses and no protection (42). Another candidate (RV3) also elicited relatively poor immune responses and marginal protection in initial trials but is still under development (43). Two neonatal strains isolated in India (116E and I321) were tested for immunogenicity in infants but only 116E elicited substantial immune responses and is being further developed as a vaccine candidate (44).

The mechanisms of protection against rotavirus disease following a live virus infection have been examined. However, the outcome has left open a crucial question, that is, is neutralizing antibody (NA) the only significant effector of protection after live rotavirus infection? Lack of a definitive answer has led to two distinctly different approaches in the development of live rotavirus vaccine candidates. If NA is essential for protection, a live rotavirus vaccine should contain strains of rotavirus with serotype-specific epitopes to protect against rotaviruses belonging to the different serotypes. If, on the other hand, effectors other than classical NA are important, vaccination with a single strain of rotavirus may be sufficient to protect against multiple serotypes. The vaccine candidates today are outcomes of these contrasting opinions. Studies that support each approach will be presented below along with the results of efficacy trials with vaccine candidates exploiting each approach.

EVIDENCE THAT NEUTRALIZING ANTIBODY IS AN ESSENTIAL EFFECTOR OF PROTECTION AGAINST ROTAVIRUS DISEASE AFER LIVE ROTAVIRUS INFECTION OF HUMANS Natural infection or immunization with live rotaviruses induces a broad range of circulating and secretory antibody responses as well as cell-mediated immune responses (49).

Table 2 Live Rotavirus Vaccine Candidates That Have Been Evaluated in Clinical Trials Vaccine candidate

Type of vaccine

Status of vaccine

RIT 4237 WC3 RRV LLR M37 RV3 I321 116E Rotarix RRV-TV (RotashieldTM) WC3-QV (RotaTeqTM) U.K. bovine/human

Single strain bovine virus Single strain bovine virus Single strain simian virus Single strain lamb virus Single strain neonatal virus Single strain neonatal virus Single strain neonatal virus Single strain neonatal virus Single strain infant virus Tetravalent reassortant viruses Pentavalent reassortant viruses Multivalent reassortant viruses

Discontinued Developed into reassortants Developed into reassortants Being sold in China Discontinued Being evaluated Discontinued Being evaluated Licensed in >110 countries Withdrawn from U.S. market Licensed in multiple countries Being evaluated

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Attempts to correlate levels of serotype-specific serum NA with protection following immunization have failed. However, evidence for the role of serotype-specificity in protection against rotavirus disease has been derived from epidemiologic evidence, vaccination/challenge studies in animals, and experimental vaccination of infants. A single rotavirus infection typically protects against subsequent severe rotavirus illnesses (4). This suggests that these infections provide protection against all rotavirus serotypes. However, severe rotavirus disease has been recorded in subjects with demonstrable serologic evidence of prior rotavirus infection (50–52). While second episodes of severe human disease caused by the same serotype have been reported, sequential rotavirus illnesses have more often been associated with different serotypes (4,53). Thus, epidemiologic evidence for the absolute importance of NA is equivocal. Differences in conclusions obtained from vaccination/ challenge experiments in animals have also never been resolved. Early studies revealed cross-protection between simian and murine rotavirus serotypes in mice (54,55). Other studies showed cross-protection between bovine rotavirus and human serotype rotavirus in calves (56) and piglets (57). The latter supported the use of bovine rotavirus in the first extensive vaccination studies in infants. However, contrasting evidence was also obtained in some vaccination/challenge studies in animals. Some crossprotection studies with distinct bovine serotypes in calves and porcine serotypes in swine revealed only serotype-specific immune protection (58,59); other studies in these domestic animals support the opposite conclusion (60,61). The most important evidence of a role for NA in protection against rotavirus is that obtained from clinical trials in infants. The first trials were performed with bovine strain RIT 4237, which is serotypically unrelated to human rotaviruses. This vaccine provided protection against human rotavirus disease in several early trials but failed in later trials (29–32). Subsequently, trials with another bovine rotavirus strain (WC3) also showed inconsistent protection (33–35). Other clinical trials were conducted in infants with RRV, a simian strain related to a common human rotavirus serotype (G3). RRV most consistently protected in trials where the natural challenge was predominately G3 (36–39). The common experience with these three animal rotavirus vaccines was that they often protected against heterotypic human rotaviruses, but their inconsistency rendered them inadequate for universal application. Recognizing their apparent clinical safety, attempts were made to enhance the protective efficacy of animal rotaviruses by incorporation of gene segments encoding neutralization proteins of common human rotavirus serotypes. The resulting multivalent ‘‘reassortant’’ vaccines have been consistently protective in clinical trials. Therefore, empirical evidence that vaccine candidates of animal rotavirus origin need to contain neutralization protein genes of common human serotypes to provide consistent protection represents the most definitive evidence for the importance of NA in protection.

LIVE ROTAVIRUS VACCINE CANDIDATES DEVELOPED EXPLOITING EVIDENCE THAT NEUTRALIZING ANTIBODY IS THE ESSENTIAL EFFECTOR OF PROTECTION The first multivalent reassortant vaccine was developed from the G3 simian RRV strain. RRV reassortants containing VP7 genes from G1, G2, and G4 human rotaviruses were generated

and these reassortants, along with RRV, were combined into the tetravalent RRV (RRV-TV) vaccine later called Rotashield. This vaccine candidate was found to consistently elicit approximately 50% protection against all rotavirus disease and more than 70% protection against severe disease (36,62–64). RRV-TV was licensed and marketed in the United States beginning in 1998 with a recommendation for universal use. Unfortunately, post-licensure surveillance indicated an association between RRV-TV vaccination and IS, and the vaccine was withdrawn (40). Multivalent reassortant vaccines were also developed between bovine and human rotaviruses. The first was with the bovine strain WC3, which became the backbone for the pentavalent RotaTeq vaccine that contained reassortants with serotype G1, G2, G3, G4, and P1a specificities. After a series of small trials with this candidate vaccine, a large trial was conducted, primarily in Finland and the United States, with more than 70,000 infants (65). The vaccine was found to be safe and induced no fever, but, most importantly, there was no association with IS. RotaTeq was also highly effective, reducing all cases of G1 to G4 rotavirus gastroenteritis by 74.0%, severe gastroenteritis by 98.0%, and hospitalizations and emergency room visits by 94.5%. Interestingly, the efficacy of the vaccine against all gastroenteritis-related hospitalizations after the first dose was 58.9%, a number that exceeded the expected fraction because of rotavirus. This vaccine was licensed in the United States in 2006 and has been recommended by the Advisory Committee on Immunization Practices (ACIP) for routine immunization of all U.S. children. Another bovine/human reassortant vaccine was also developed and has been evaluated in two small trials (66,67). This vaccine is based on the U.K. strain of bovine rotavirus and contains VP7 genes from G1, G2, G3, or G4 human rotaviruses. Protection was significant (60% against any rotavirus disease and 90% against severe rotavirus disease; P < 0.02). To facilitate the commercialization of this vaccine, the NIH Office of Technology Transfer has granted licenses to manufacturers around the world, primarily in developing nations.

EVIDENCE THAT NEUTRALIZING ANTIBODY IS NOT AN ESSENTIAL EFFECTOR OF PROTECTION AFTER LIVE ROTAVIRUS INFECTION OF HUMANS Evidence that NA is not the only product of the adaptive immune system responsible for prevention of rotavirus disease has been provided from two types of studies. These include studies on immunity after natural or experimental rotavirus infection of either animals or humans and studies associated with vaccination of humans with live rotaviruses. Some of the initial studies on experimental infection of piglets and calves suggested that NA was a critical component of protection against disease following subsequent rotavirus challenge (58,59,68) but later studies suggested the opposite (60,61). Studies in naturally infected humans have also suggested that NA is not the only effector of protection. Neonatal rotavirus infections have protected against subsequent rotavirus disease even when their serotypes were distinct from the circulating human rotaviruses (26–28). Rotavirus infections in older infants have also provided substantial protection against subsequent rotavirus disease, particularly severe disease, and sequential illnesses that did occur were sometimes caused by the same rotavirus serotypes. In a study with Bangladeshi children, only NA titers to heterotypic rotaviruses were found to be

Chapter 53: Vaccines Against Rotavirus Gastroenteritis

independently associated with protection against rotavirus disease (50). In a Mexican study, repetitive rotavirus infections were found to occur between birth and two years of age, but no moderate-to-severe rotavirus illnesses were found after the second infection even though the four major G serotypes of human rotavirus were co-circulating (4). These results can be interpreted to suggest that protection, at least against more severe rotavirus disease, was not strictly serotype-specific. Studies on vaccination of humans with live rotaviruses have provided mixed signals regarding the importance of NA. Both bovine strains RIT 4237 and WC3 were efficacious when first evaluated even though they were serotypically unrelated to circulating human rotavirus strains. Both, however, failed when evaluated in developing countries as well as in some studies in developed countries. Similar inconsistencies were found with the monovalent RRV vaccine. When RRV and WC3 were developed into reassortant vaccines, NA responses to human G types represented in these vaccines were sometimes found in only a small percentage of vaccines, and no correlation has been found between titers of serum NA to specific G types and protection (69). Even so, both vaccines have consistently elicited more than 70% protection against severe rotavirus disease.

LIVE ROTAVIRUS VACCINE CANDIDATES DEVELOPED FROM EVIDENCE THAT NEUTRALIZING ANTIBODY IS NOT ESSENTIAL FOR PROTECTION B and T cell epitopes responsible for stimulating protective immune responses after vaccination could potentially reside on any rotavirus protein, and these epitopes may be conserved within different rotavirus serotypes. This led to the development of several vaccine candidates consisting of single rotavirus strains. Although development of several monovalent candidates has been discontinued because of inconsistent efficacies, evaluation of others is continuing. These include one animal (lamb) strain, two neonatal strains, and one strain obtained from a symptomatically infected child. The lamb strain is being marketed in China although its efficacy has not been established. The two neonatal strains (RV3 and 116E) are in the early stages of development. However, the strain from the symptomatic child, attenuated by multiple cell culture passages, is being licensed worldwide today as Rotarix. Initial safety testing revealed the Rotarix vaccine was safe and did not induce fever as seen following vaccination with its 89-12 parent (47). Subsequent reports from Singapore (70), Finland (66), Latin America (48,71), and the United States (72) verified the vaccine was not associated with fever but remained highly immunogenic. The vaccine also did not interfere with the immune responses to concomitantly used vaccines including oral polio vaccine (OPV) (71,72). In an initial efficacy trial in Finland conducted over two rotavirus seasons, the vaccine was 73% protective against all rotavirus gastroenteritis and 90% protective against severe gastroenteritis (66). In a trial of more than 63,000 infants conducted primarily in several countries in Latin America, Rotarix did not induce fever and, most importantly, was not associated with IS (48). In this large study, efficacy was 85% against severe rotavirus diarrhea and hospitalizations, and reached 100% against more severe gastroenteritis. Of note, efficacy was high (over 86%) against severe rotavirus diarrhea caused not only by G1P(8) strains but also by the VP4 related G3P(8), G4P(8), and G9P(8)

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strains. In the most recent trial of more than 4000 infants conducted in six European countries, protection reached 100% against hospitalization due to rotavirus (73). In this study, efficacy against severe disease due to G3P(8), G4P(8), and G9P(8) strains was again similar to that against G1P(8) strains and exceeded 95%, while efficacy against heterotypic G2P(4) strains was 75%. Efficacy against hospitalization due to gastroenteritis of any cause was also 75%. The Rotarix vaccine was originally licensed in Mexico in 2004 and has since been licensed in more than 90 countries. It is being administered as part of the routine childhood immunization series in several nations, particularly in Latin America, and is being sold on the private market in most of the other countries where it is licensed.

VACCINE STRATEGIES NOT UTILIZING LIVE ROTAVIRUSES A successful live rotavirus vaccine should consistently protect against severe rotavirus illnesses. This goal has been realized, where tested, for the two rotavirus vaccines now being licensed worldwide and for Rotashield. However, no efficacy trials with these candidates have been completed in third world nations where rotavirus deaths are most common and earlier rotavirus vaccine candidates failed. On the basis of lingering concerns with live rotavirus vaccines, nonliving vaccines are being developed. Three types have been given the most attention. These include inactivated triple- and double-layered (lacking VP4 and VP7) rotavirus particles, triple- and double-layered virus-like particles (VLPs), and recombinant, expressed VP6 proteins. Candidates representative of each have been under development and tested in animal models but not in humans. Vaccination with inactivated rotavirus particles delivered by either parenteral or mucosal routes blocked intestinal rotavirus replication in adult mice (74,75). Triple-layered particles were more effective than double-layered particles only when their serotype matched that of the challenge virus. This suggested that neutralizing antibodies played a role in protection but were not required. Studies in gnotobiotic piglets indicated that protection after either oral or intramuscular delivery of inactivated viruses was significantly less effective than found after oral vaccination with live virus (76,77). However, protection in this piglet model is typically less effective than in adult mice. It remains to be determined which model is more applicable to humans. VLP vaccines can contain only VP2 and VP6 (2/6 VLPs) or additionally incorporate one or more of the neutralization proteins, VP4 and VP7 (2/6/4/7 VLPs). Most recent studies have utilized 2/6 VLPs administered with effective adjuvants. Both intranasal and intrarectal immunization of mice with these particles has resulted in excellent protection against fecal rotavirus shedding following murine rotavirus challenge (78–81). VLP rotavirus vaccines have also been delivered intramuscularly to both mice and rabbits where they produced effective protection against fecal shedding (82,83). In contrast, gnotobiotic piglets intranasally immunized with 2/6 VLPs and adjuvant were neither protected against fecal shedding of the challenge virus nor diarrheal illness (84). Vaccine candidates composed only of chimeric, Escherichia coli–expressed VP6 proteins from murine or human rotaviruses have also been found to protect mice against fecal rotavirus shedding when administered either intranasally, orally, or intrarectally with effective adjuvants (85–87). The level of protection

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has been consistently more than 90% by any of these routes and protection was found to remain fully intact for at least one year. VP6 is the group antigen and, therefore, is highly conserved within group A rotaviruses. Accordingly, protection elicited by intranasal immunization with a human rotavirus VP6 protein was also highly protective against fecal rotavirus shedding following challenge with heterotypic murine rotaviruses (86). All three types of nonliving rotavirus vaccine candidates have been under development for years, but the successes of the live candidates have stifled commercial interest in these products. As long as there is not another incident such as that which occurred with Rotashield to shatter public confidence in the live candidates, evaluation of any of the nonliving candidates in human trials is expected to continue to be delayed.

FUTURE CONSIDERATIONS The abrupt and unanticipated withdrawal of the Rotashield vaccine within a year of its introduction into the U.S. childhood immunization program in 1998 was a major setback to international efforts to develop rotavirus vaccines. Furthermore, the lack of a clear mechanism for the association between Rotashield and IS raised questions about the safety of other live oral rotavirus vaccines. Manufacturers of candidate vaccines were faced with the daunting prospect of undertaking large and expensive pre-licensure trials to demonstrate that their products did not carry a risk of IS. It is remarkable, therefore, that within seven years of the debacle with Rotashield, two new rotavirus vaccines are now licensed and being introduced into immunization programs worldwide. RotaTeq has been recommended for routine use in all U.S. infants and is also licensed in many other countries. In October 2006, through a manufacturer sponsored vaccine donation program of three-year duration, RotaTeq was launched nationwide for immunization of infants in Nicaragua. Rotarix was licensed in the USA in 2008 and is being used in national immunization programs in several Latin American countries, including Brazil, Mexico, Venezuela, Panama, and El Salvador. Rotarix is also licensed in more than 110 countries globally, including those of the European Union. In April 2009, the Global Alliance for Vaccines and Immunizations (GAVI) approved the use of public sector funds to purchase Rotarix in eligible countries worldwide. Despite these exciting developments, several challenges remain before the full potential of rotavirus vaccines can be realized. In light of the experience with the previous Rotashield vaccine, concerns remain about the possible association of current rotavirus vaccines with IS at a level of risk that would not have been detected even in the large pre-licensure trials. Thus, post-licensure safety surveillance is a high priority. In the United States, cases of IS have been reported through a national passive surveillance system within the first seven days after vaccination with RotaTeq. However, on the basis of estimates that over 10 million doses of RotaTeq have been distributed in the United States, the reported number of IS cases did not exceed the background number expected. Although these data suggest that RotaTeq is not associated with an increased risk of IS, it is never possible to fully exclude that either this or other rotavirus vaccines will not have some associated increased risk of IS or another adverse event. Another key issue that remains to be addressed is whether these live, oral rotavirus vaccines will be efficacious among

children in developing countries where they are most needed. Other live oral vaccines—OPV, cholera vaccines, and even previous rotavirus vaccines—that have worked well in affluent countries have sometimes failed in trials in developing countries. The performance of oral rotavirus vaccines could be impaired in developing countries by factors such as malnutrition, colonization of the gut with interfering microbes, interference by maternal antibodies, and frequent existence of comorbidities like malaria. Thus, clinical trials will be required to demonstrate the performance of these vaccines in developing countries. Trials of both Rotarix and RotaTeq in Africa and Asia are ongoing and results are anticipated in 2009 to 2010. If rotavirus vaccines are determined to be safe and efficacious for use globally, their introduction into immunization programs will likely require substantial input from the international donor community, such as the GAVI Alliance (88) and the Bill & Melinda Gates Foundation (89). GAVI has already approved purchase of rotavirus vaccines for eligible countries in Latin America and Europe at a cost to the country of US$0.15 to US$0.20 per vaccine dose. In the long term, the production of vaccines by vaccine makers in China, India, and Indonesia, who are currently developing rotavirus vaccines could provide an additional supply of vaccine, and the increased competition could reduce the cost of vaccines. Ensuring that rotavirus vaccines reach the poorest children in the world who are at greatest risk of severe rotavirus disease will be critical to realize their full life saving potential.

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54 Novel Strategies for Immunizing Infants in Developing Countries Who Are Too Young to Receive the Currently Licensed Measles Vaccines Marcela F. Pasetti, Karina Ramirez, Karen L. Kotloff, Eileen M. Barry, and Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

MEASLES IN DEVELOPING COUNTRIES In 1999 the World Health Organization (WHO) reported that measles was the third most important cause of mortality among children less than five years of age in developing countries (1), with most deaths occurring among children living in certain areas of the Indian subcontinent and in sub-Saharan Africa (2). This disease burden persisted despite the existence of highly efficacious attenuated measles virus (MV) vaccines that are recommended to be given routinely through the Expanded Programme on Immunization (EPI) to infants *9 months of age living in developing countries. A limitation of the current licensed live attenuated measles vaccine is that, although efficacious in older infants, children, and adults, it fails to reliably immunize young infants (<6 months of age) because of the presence of maternal antibodies and the immaturity of their immune system. Importantly, a notable proportion of measles deaths in developing countries occur among young infants during the so-called ‘‘window of vulnerability,’’ spanning approximately four to nine months of age (3). During this period, titers of maternally derived measles antibodies drop to a level that cannot provide protection against clinically apparent infection if infants are exposed to wild-type measles (4). Nevertheless, these low antibody titers, in conjunction with the infant’s immature immune status can interfere with successful immunization using the available attenuated measles vaccine. Consequently, severe clinical measles can ensue when such young infants are exposed to fully virulent wild-type MV (5,6). Case fatality rates from measles are inordinately high (*10– 25%) during these early months of life in developing country venues (7,8). Considerable progress has been made since 2000 in diminishing mortality from measles, including young infants, by improving routine vaccine coverage with the first dose along with providing a second opportunity for measles vaccination, most often by undertaking mass national and subnational immunization campaigns (9,10). Young infants are indirectly protected if mass campaigns achieve high levels of coverage

that diminishes the transmission of wild-type virus in the community. However, the high levels of coverage (>90%) with two doses of measles vaccine that are considered necessary to maintain the current reductions in measles mortality remain a difficult goal to sustain (11). An estimated 242,000 deaths from measles occurred in 2006, mostly among children living in Southeast Asia and sub-Saharan Africa (10). The continuing high case fatality rate among children under five years of age with poor access to appropriate treatment was recently illustrated in several countries in sub-Saharan Africa with low immunization coverage (12). In that study, the highest case fatality rates were almost always among infants younger than 12 months of age. The extreme transmissibility of MV, one of the most contagious of all human viral pathogens, requires that high vaccine coverage rate (and population immunity) be maintained lest susceptibles accumulate in sufficient numbers for the chain of facile transmission to recur should the virus be introduced into a population. Indeed, outbreaks can occur in population groups that have fewer than 10% susceptible individuals. Currently, the herd immunity that results from high coverage rates among individuals 10 months of age and older is the only mechanism available for protecting the most vulnerable hosts (young infants). Thus, having a means to directly protect infants who are otherwise too young to respond reliably to the current licensed measles vaccines would provide a useful adjunct for measles control.

APPROACHES FOR PROTECTING YOUNG INFANTS AGAINST MEASLES Three main approaches to immunizing young infants during the window of vulnerability have been assessed. One strategy evaluated a 100-fold higher than usual dose of vaccine (13). A second approach explored specific strains of attenuated MV [e.g., Edmonston-Zagreb (EZ) strain] (14,15). The third strategy involved aerosol administration of live measles vaccine (16). None of these approaches was adopted for large-scale intervention. The first strategy was abandoned for lack of safety after clinical trials in

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several developing countries showed a poorly understood but significant increase in overall mortality among girls who, as young infants, received the high dose compared with the standard dose of vaccine (13). The second approach was discarded when no specific attenuated strain proved to be markedly superior to other currently licensed strains (15). The aerosol approach has shown some promise for older infants and children, but for young infants, it is compromised by the lack of a practical and efficient method of administering aerosolized vaccine and also by inconsistent results (16) that sometimes revealed lower immunogenicity compared with subcutaneous immunization (17,18). The phenomenon of herd immunity has been invoked as a strategy for protecting young infants during the window of vulnerability age interval. It is expected that as immunization coverage increases in a community, the risk of measles exposure before the age of nine months will diminish. However, this strategy has not always been effective. In urban districts of Guinea-Bissau, an increase in vaccine coverage from 61% to 80% did not reduce measles incidence among infants <9 months of age, presumably because of the virus’ extreme contagiousness (19). If crowding is present, even if coverage is very high, MV manages to infect the susceptibles (20). Furthermore, although the recent experience of the measles elimination program in the Americas shows that transmission can be interrupted, sustained control of endemic measles requires that very high immunization coverage be maintained. This has necessitated a first dose at 9 to 12 months or shortly thereafter, plus a second dose provided through either routine services and/or repeated supplemental campaigns (21). The occurrence of multi-country outbreaks involving tens of thousands of cases in Latin America (following an importation) illustrates the daunting task of sustaining measles elimination despite the implementation of supplemental campaigns (22). Currently, all countries in the Americas, and selected countries in Europe, the Middle East, sub-Saharan Africa, Oceania, and Asia have adopted immunization strategies aimed at measles elimination and have made substantial progress toward this goal (10,23). These successes have led some experts to believe that measles elimination in all regions of the world is feasible on the basis of existing measles vaccine and strategies (24). However, other authorities, taking into account the extreme transmissibility of measles and the limitations of the current vaccines, are more skeptical (25). Still others take the view that measles elimination in some regions such as sub-Saharan Africa might be possible if an improved vaccine were available that could reliably immunize and protect very young infants (26). The following characteristics would be desirable for an improved, new measles vaccine: (i) is safe for young infants; (ii) is not neutralized by maternal antibody; (iii) induces protective immunity equivalent to the current vaccines; (iv) is effective earlier in life than the current vaccines; (v) is amenable to large-scale economical manufacture; and (vi) is easy to administer. Optimally, the vaccine could be administered using a needle-free delivery system.

IMMUNOLOGICAL CORRELATES OF PROTECTION AGAINST MEASLES Humoral immunity is important in preventing viral entry into cells that could initiate infection. This is clearly shown by the protection conferred to newborns by maternal antibodies and the efficacy of postexposure administration of measles immune globulins to susceptible individuals. The strongest correlate of protection against measles disease is the presence of plaque reduction neutralization (PRN) serum antibodies; a titer
1:120

(or
200 mIU/mL) has been associated with clinical protection (27). Studies in rhesus macaques have shown that measles antibodies of high avidity and neutralization capacity are required to avoid occurrence of the atypical measles syndrome upon exposure to wild-type MV, as was seen in recipients of formalin-inactivated measles vaccine (28). IgG avidity has been used as a tool to determine vaccine failure, and in such cases, revaccination could have prevented disease (29). It is unclear, however, whether antibodies have a role in virus clearance after infection is initiated (30). Cell-mediated immunity (CMI), and particularly CD8þ cytotoxic T lymphocytes (CTL) and CD4þ Th1-type cells appear to play a critical role in recovery from illness by controlling viral replication (30). Agammaglobulinemic patients can recover normally from measles, indicating that CMI alone can be effective in the absence of antibodies (31). In contrast, mortality in patients with T-cell deficiencies (e.g., HIV) can reach 50% to 100% (32). The contribution of CMI to preventing measles infection in humans in the light of ‘‘failed’’ seroconversion after vaccination has been increasingly recognized (33–37). Studies in animal models provide additional evidence that nonhumoral responses may contribute to protection. Rhesus macaques immunized with a measles DNA vaccine in the presence of passively transferred measles antibodies elicited weak or absent serological responses but were nonetheless protected from MV challenge (38). Rhesus macaques depleted of CD20þ B cells (by infusion of Mab prior to challenge) exhibited delayed antibody responses but unaltered kinetics of virus clearance, whereas those depleted of CD20þ and CD8þ, or CD8þ T cells alone had prolonged viremia and more severe rash (39,40). Measles DNA vaccination of newborn monkeys in the presence of passively transferred measles immunoglobulins (to mimic maternal antibodies) elicited CMI that reduced postchallenge viremia (41) and conferred 60% to 80% protection when an IL-2 molecular adjuvant was given 48 hours later (41) or at the time of vaccination (42). These animals, however, did not develop neutralizing antibodies (42). Measles infection has long been associated with immunosuppression, characterized by inhibition of T-cell proliferation, impaired antigen presentation and cytotoxic function, reduced B lymphocyte maturation and antibody production, and switch from T h 1- to T h 2-type cytokine polarization (30,43,44). The mechanisms underlying these effects are poorly understood. Therefore, any new measles vaccine mimicking virus infection or containing viral structural proteins must also be carefully investigated to rule out potential inhibition of T cell responses or any other form of immunosuppression.

MEASLES DNA VACCINES FOR PRIMING THE VERY YOUNG INFANTS’ IMMUNE SYSTEM TO RESPOND RELIABLY TO CURRENT LICENSED MEALSES VACCINE Because of their capacity to produce vaccine antigens in an intracellular niche, DNA vaccines offer a promising means of priming young infant hosts in the face of placentally transferred maternal measles antibodies (41,42). In newborn mice, DNA vaccines elicit high-quality and long-lasting antibody responses and can overcome deficient induction of Th1 and CTL responses, enhancing the capacity of young hosts to clear intracellular pathogens (45–48). They have been used in combination with other vaccine delivery systems in ‘‘heterologous prime-boost strategies’’ to enhance protective immunity against

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infectious agents, particularly viruses and protozoa, which previously posed great problems for vaccine development (49). The prime-boost approach increases and broadens immune responses compared with a single immunization or a homologous prime boost (50), demonstrated by higher antibody levels and frequency of antigen-specific T cells, selective enrichment of high-avidity antibodies and T cells, and increased efficacy against pathogen challenge (51,52).

Development of Sindbis Replicon Measles DNA Vaccines CVD investigators developed two Sindbis replicon-based measles DNA vaccine candidates to specifically target infants who are too young to receive the current licensed measles vaccines (53–57). The aim of these measles DNA vaccines is to prime the young infant immune system to respond safely and effectively to a subsequent boost with the currently licensed attenuated measles vaccine. Sindbis replicons represent a new generation of improved DNA vaccines in which cDNAs driven by eukaryotic promoters express self-replicating replicon RNAs (58). Transcription from the cytomegalovirus (CMV) promoter within a mammalian cell gives rise to a Sindbis virus RNA replicon vector, which programs its own cytoplasmic RNA amplification and high-level expression of the heterologous measles gene (s) via the alphavirus subgenomic promoter. The increased immunogenicity of Sindbis-based DNA vaccines is not only due to increased antigen production. Cells transfected with Sindbis DNA replicons elaborate double-stranded RNA, which enhances immune responses by stimulating toll-like receptor 3 (TLR3) on antigen-presenting cells and induces various cytokines. Cells transfected with Sindbis virus–based plasmids undergo apoptotic death releasing antigenic material for cross-presentation and dsRNA, which provides additional proinflammatory immune stimulation (59,60). Furthermore, Sindbis virus–derived dsRNA can activate and enhance maturation of DC (61), a major requirement for the induction of Th1type immunity early in life (62). A modified backbone Sindbis replicon (pSINCP), developed by scientists at Chiron (now Novartis) vaccines, which incorporates nonstructural protein gene sequences from a human dendritic cell (DC)-tropic Sindbis virus, was further modified by inserting genes that encode the putative protective measles hemagglutinin (H) antigen alone or with measles fusion (F) protein (53,57). These antigens were selected for several reasons. The H protein mediates viral entry to the host cell and is the main viral antigen against which neutralizing antibodies are directed. The F protein mediates fusion of the viral envelope with the cell membrane. In animal models, PRN antibody responses induced by DNA vaccines encoding both H and F are somewhat diminished when compared with those encoding H alone (63). This is true whether the H and F vaccines are coadministered or whether a single DNA vaccine encoding both H and F is administered. On the other hand, CMI responses are typically broader if F protein as well as H are included in the DNA vaccine (57), and such responses may synergize the protective effect of neutralizing antibodies. Some measles virologists and vaccinologists believe that the safety of the vaccine may be enhanced if F as well as H antigens are included in a new measles vaccine (64). It has been

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argued that an imbalance in antibodies against H and F antigens, mainly the absence of F antibodies, was responsible for the atypical measles syndrome seen in the 1960s in children who received the formalin-inactivated measles vaccine and were later exposed to wild-type MV (65). Robust data do not exist to support this bias, and indeed, modern data generated in the rhesus challenge model refute the view (28). Polack et al. showed that juvenile monkeys immunized with measles DNA vaccines encoding H and F proteins alone or in combination mounted PRN titers and long-lasting CD8þ-mediated CTL (63); these animals were protected and did not develop atypical measles after challenge with wild-type MV (63). Finally, if one wishes to deliver both H and F genes as part of a DNA vaccine, engineering a single DNA vaccine construct is preferable to coadministering two different plasmids in terms of diminishing the complexity, logistics, cost of manufacture, and quality control of product (57). Two Sindbis replicons were produced: pMSIN-H, which contains only the H gene and directs expression of the MV H, and pMSINH-FdU, a bicistronic construct, which contains the H and the F genes and directs expression of both glycoproteins causing syncytia formation in susceptible Vero cells (53,57).

Preclinical Safety, Immunogenicity, and Efficacy Studies The Sindbis DNA replicons encoding MV antigens were administered to small animals (cotton rats and newborn and adult mice) intramuscularly (IM) by needle and syringe (53,56,57,66), to juvenile and very young infant (*45 days of age) rhesus monkeys intradermally (ID) by means of the Biojector1 2000 needle-free injection device (54), and to juvenile rhesus monkeys IM via Biojector 2000 (54). The DNA vaccines were well tolerated in all the animal species tested. They were also immunogenic in adult and in newborn mice and induced protective immunity against measles infection in cotton rats; immune responses were further increased when a two-dose DNA ID priming series was followed by IM boost with live attenuated EZ measles vaccine. In very young infant as well as in juvenile rhesus monkeys, the measles DNA vaccines elicited PRN antibodies and measlesspecific IFN-g-secreting T cells. In these immunogenicity studies in monkeys as well as in the studies in rodents, pMSIN-H stimulated stronger serum PRN responses than pMSINH-FdU. Priming with pMSIN-H succeeded in eliciting PRN titers above the protective threshold (
200 mIU/mL). It was also highly immunogenic in newborn mice in the presence of maternal antibodies, and the serological responses measured were of high avidity and neutralizing capacity. In very young infant macaques, 1.0-mg priming doses were more immunogenic than 0.5-mg doses. The ID route also proved somewhat more immunogenic for priming than the IM route. A heterologous prime-boost regimen consisting of priming with 1.0-mg ID doses of pMSIN-H or pMSINH-FdU and boosting with aerosolized attenuated MV vaccine was well tolerated by juvenile macaques and protected against disease and viremia following challenge with wild-type MV 16 months later. A prime-boost regimen consisting of priming with 1.0-mg or 0.5-mg ID doses of pMSIN-H or 0.5-mg doses of pMSINH-FdU and boosting with aerosolized attenuated MV vaccine was well tolerated by very young infant rhesus macaque monkeys and protected against viremia following challenge with wild-type

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MV nine months later. No evidence was found in these experiments of histopathological features consistent with ‘‘atypical measles’’ (63,67). Future studies of interest for these vaccines in nonhuman primates include immunogenicity in the presence of maternal antibodies and analysis of immunosuppression.

Biodistribution, Integration, and Toxicology Studies Studies assessing the biodistribution of the DNA vaccines were undertaken to detect any evidence of DNA integration (55). pMSIN-H and pMSINH-FdU were administered ID to New Zealand white rabbits at their intended clinical dosage levels via the Biojector 2000 needle-free injection system and biodistribution was monitored during a 60-day period. A single dose of 1.76 mg of pMSIN-H or 1.84 mg of pMSINH-FdU had no effect on mortality, clinical and cageside observations, body weights, body weight changes, and food consumption. The only vaccine-related effects observed were minimal transient erythema, edema, and inflammation confined to the injection site. The plasmids persisted at the injection site skin and subcutis, injection site muscle, and (to a much lesser degree) in the popliteal lymph nodes that drain the injection sites for the duration of the study (55). Integration studies showed no evidence of plasmid integration into the rabbit host genome. To assess potential toxicological effects, New Zealand white rabbits were primed ID with pMSIN-H (1.76 mg), pMSINH-FdU (1.84 mg), or phosphate buffered saline (PBS) (control article), on study days 1, 29, and 57 (55). Some animals received a subcutaneous (SC) injection (boost) of 0.5 ml of PBS or *103 tissue culture 50% infective dose (TCID50) of the EZ measles vaccine. Subgroups were euthanized at different time points prior to and after the boost, and no effects were found on mortality, clinical observations, cageside observations, body weights, body weight changes, food consumption, clinical pathology, organ weights, or organ weight ratios. Increased frequency, score, and recovery time of dermal Draize observations at the pMSIN-H, and pMSINH-FdU injection sites were observed, which correlated with injection site gross findings (red discoloration on study day 60 only) and histopathological findings of inflammation that recovered with time (55). Both Sindbis-based vaccine plasmids were immunogenic in rabbits; as observed in other species, pMSIN-H elicited higher PRN titers.

Phase I Clinical Studies The extensive preclinical data demonstrating the safety, immunogenicity, and efficacy of the Sindbis replicon measles vaccines led to filing of a new investigational drug application (IND) to support the performance of a phase I clinical trial. On the basis of the superior immunogenicity and efficacy of pMSIN-H in the extensive preclinical animal model experiments, it was the favored DNA vaccine candidate to move forward in clinical trials. Nevertheless, on the assumption that humans might respond differently, we elected also to study pMSINH-FdU vaccine, in addition to pMSIN-H, at least in phase I. We undertook conduct of the phase I trial of the DNA vaccines in healthy adults of ages 18 to 45 years living in the United States who participated in a randomized, double-blind, placebo-controlled, dose-escalating, outpatient study to assess three dosage levels of approximately 200, 400, and 800 mg of each vaccine in a stepwise fashion. At each dosage level, 20 subjects were allocated to one of four groups to receive

two doses of vaccine and one dose of placebo on days 0, 28, and 56, as follows: (i) pMSIN-H, pMSIN-H, placebo (n ¼ 5); (ii) placebo, pMSIN-H, pMSIN-H (n ¼ 5); (iii) pMSINH-FdU, pMSINH-FdU, placebo (n ¼ 5); or (iv) placebo, pMSINH-FdU, pMSINH-FdU(n ¼ 5). The vaccines were administered ID using Biojector 2000 (Kotloff and Levine, personal communication). The purpose of this study was to generate preliminary safety data prior to considering the evaluation of the safety and immunogenicity of this regimen in developing countries, where the ultimate target population resides. Since routine infant immunization in sub-Saharan Africa involves contacts at 6, 10, and 14 weeks of age, the ultimate goal is to administer one of these DNA vaccines at 6 and 10 weeks of age as the priming immunogen, followed by a dose of currently licensed attenuated measles vaccine as the boosting immunogen at 14 weeks of age. This strategy, if successful, would allow an infant to be immunized before the window of vulnerability opens at *16 weeks of age.

ALTERNATIVE MEASLES VACCINE CANDIDATES A handful of DNA vectors have been proposed as potential measles vaccine candidates and shown to prime immune responses in juvenile (63,68,69) and very young infant macaques (41,42) and to confer varying degrees of protection against wild-type MV challenge. Immune responses, however, were quite variable. When administered to infant macaques, they either failed to develop neutralizing antibodies (42) or generated poor responses (70). Different approaches have been described to improve the immunogenicity of these vaccines including the use of codon-optimized genes and adjuvants such as vaxfectin (70) and plasmid-encoded cytokines (42). Other genetic vaccine candidates explored include recombinant viral vectors such as alphavirus replicon particles (71), parainfluenza (72), and vaccinia virus expressing MV antigens (69,73,74). Subunit and epitope-based vaccines have been described (75,76), but they elicit limited and short-term immunity. A proteosome-MV H and F vaccine administered to juvenile rhesus macaques alone in three consecutive immunizations or as a boost following priming with Sindbis virus measles DNA vaccines conferred full protection against MV challenge (54). Despite their initial promise in preclinical studies, none of these approaches has yet been investigated in humans.

CONCLUSION Despite the overall progress achieved with mass immunization campaigns, in several countries in sub-Saharan Africa (e.g., Niger, Chad), measles mortality in young children remains a serious health problem (12). A Sindbis replicon measles DNA vaccine encoding measles H was shown to be highly immunogenic and to induce protective immunity in nonhuman primates. Among alternative vaccine strategies for young infants, this is the most advanced, having been tested in a phase I study that is nearing completion. The ultimate goal is to administer such a new measles vaccine candidate at 6 and 10 weeks of age as the priming immunogen, followed by the currently licensed attenuated measles vaccine as the boosting immunogen at 14 weeks of age. This strategy, if successful, would provide a means to protect infants during the critical window of vulnerability.

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45. Hassett DE, Zhang J, Slifka M, et al. Immune responses following neonatal DNA vaccination are long-lived, abundant, and qualitatively similar to those induced by conventional immunization. J Virol 2000; 74(6):2620–2627. 46. Hassett DE, Zhang J, Whitton JL. Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge. J Virol 1997; 71(10):7881–7888. 47. Zhang J, Silvestri N, Whitton JL, et al. Neonates mount robust and protective adult-like CD8(þ)-T-cell responses to DNA vaccines. J Virol 2002; 76(23):11911–11919. 48. Martinez X, Brandt C, Saddallah F, et al. DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life. Proc Natl Acad Sci U S A 1997; 94(16):8726–8731. 49. Woodland DL. Jump-starting the immune system: prime-boosting comes of age. Trends Immunol 2004; 25(2):98–104. 50. McConkey SJ, Reece WH, Moorthy VS, et al. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med 2003; 9(6):729–735. 51. Ramsay AJ, Leong KH, Ramshaw IA. DNA vaccination against virus infection and enhancement of antiviral immunity following consecutive immunization with DNA and viral vectors. Immunol Cell Biol 1997; 75(4):382–388. 52. Kent SJ, Zhao A, Best SJ, et al. Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J Virol 1998; 72(12): 10180–10188. 53. Pasetti MF, Barry EM, Losonsky G, et al. Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats. J Virol 2003; 77(9):5209–5217. 54. Pasetti MF, Resendiz-Albor A, Ramirez K, et al. Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques. Clin Pharmacol Ther 2007; 82(6):672–685. 55. Ramirez K, Barry EM, Ulmer J, et al. Preclinical safety and biodistribution of Sindbis virus measles DNA vaccines administered as a single dose or followed by live attenuated measles vaccine in a heterologous prime-boost regimen. Hum Gene Ther 2008; 19(5):522–531. 56. Capozzo AV, Ramirez K, Polo JM, et al. Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies. J Immunol 2006; 176(9):5671–5681. 57. Song MK, Vindurampulle CJ, Capozzo AV, et al. Characterization of immune responses induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins. J Virol 2005; 79(15):9854–9861. 58. Hariharan MJ, Driver DA, Townsend K, et al. DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector. J Virol 1998; 72(2):950–958. 59. Leitner WW, Hwang LN, deVeer MJ, et al. Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat Med 2003; 9(1):33–39. 60. Leitner WW, Hwang LN, Bergmann-Leitner ES, et al. Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines. Vaccine 2004; 22(11–12):1537–1544.

61. Cella M, Salio M, Sakakibara Y, et al. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med 1999; 189(5):821–829. 62. Adkins B, Leclerc C, Marshall-Clarke S. Neonatal adaptive immunity comes of age. Nat Rev Immunol 2004; 4(7):553–564. 63. Polack FP, Lee SH, Permar S, et al. Successful DNA immunization against measles: neutralizing antibody against either the hemagglutinin or fusion glycoprotein protects rhesus macaques without evidence of atypical measles. Nat Med 2000; 6(7):776–781. 64. Schlereth B, Germann PG, ter Meulen V, et al. DNA vaccination with both the haemagglutinin and fusion proteins but not the nucleocapsid protein protects against experimental measles virus infection. J Gen Virol 2000; 81(pt 5):1321–1325. 65. Merz DC, Scheid A, Choppin PW. Importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection. J Exp Med 1980; 151(2):275–288. 66. Pasetti MF, Ramirez K, Resendiz-Albor A, et al. Sindbis virusbased measles DNA vaccines protect cotton rats against respiratory measles: relevance of antibodies, mucosal and systemic antibody-secreting cells, memory B cells, and Th1-type cytokines as correlates of immunity. J Virol 2009; 83(6):2789–2794. 67. Polack FP, Auwaerter PG, Lee SH, et al. Production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody. Nat Med 1999; 5(6):629–634. 68. Premenko-Lanier M, Rota P, Rhodes G, et al. Prior DNA vaccination does not interfere with the live-attenuated measles vaccine. Vaccine 2004; 22(5–6):762–765. 69. Stittelaar KJ, Wyatt LS, de Swart RL, et al. Protective immunity in macaques vaccinated with a modified vaccinia virus Ankarabased measles virus vaccine in the presence of passively acquired antibodies. J Virol 2000; 74(9):4236–4243. 70. Pan CH, Jimenez GS, Nair N, et al. Use of Vaxfectin adjuvant with DNA vaccine encoding the measles virus hemagglutinin and fusion proteins protects juvenile and infant rhesus macaques against measles virus. Clin Vaccine Immunol 2008; 15(8): 1214–1221. 71. Pan CH, Valsamakis A, Colella T, et al. Inaugural article: modulation of disease, T cell responses, and measles virus clearance in monkeys vaccinated with H-encoding alphavirus replicon particles. Proc Natl Acad Sci U S A 2005; 102(33):11581–11588. 72. Skiadopoulos MH, Surman SR, Riggs JM, et al. A chimeric humanbovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys. J Virol 2001; 75(21):10498–10504. 73. Kovarik J, Gaillard M, Martinez X, et al. Induction of adult-like antibody, Th1, and CTL responses to measles hemagglutinin by early life murine immunization with an attenuated vacciniaderived NYVAC(K1L) viral vector. Virology 2001; 285(1):12–20. 74. Zhu Y, Rota P, Wyatt L, et al. Evaluation of recombinant vaccinia virus–measles vaccines in infant rhesus macaques with preexisting measles antibody. Virology 2000; 276(1):202–213. 75. Osterhaus A, van Amerongen G, van Binnendijk R. Vaccine strategies to overcome maternal antibody mediated inhibition of measles vaccine. Vaccine 1998; 16(14–15):1479–1481. 76. El Kasmi KC, Muller CP. New strategies for closing the gap of measles susceptibility in infants: towards vaccines compatible with current vaccination schedules. Vaccine 2001; 19(17–19): 2238–2244.

55 Challenges and Prospects for the Development of an HIV Vaccine Barney S. Graham and Gary J. Nabel Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

CHALLENGES AND PROSPECTS FOR VACCINEINDUCED PROTECTION AGAINST HIV Human immunodeficiency virus type 1 (HIV-1) has caused a devastating and persistent global epidemic with profound consequences on human health and socioeconomic stability. The greatest burden of disease occurs in resource-poor settings where education, diagnosis, care, and treatment are not readily available. Each day approximately 7,000 persons are infected by HIV-1, including approximately 1,000 persons per day under the age of 15 years. The development of a vaccine would have a major impact on the spread of this disease, either by preventing infection and/or reducing viremia to slow the rate of disease progression and transmission. It remains an urgent public health need.

Desirable Characteristics of an HIV Vaccine To control the HIV epidemic optimally, a vaccine must (i) be safe and relevant for susceptible populations, (ii) work against diverse subtypes of the virus, (iii) induce durable immunity that prevents infection or contains virus, and (iv) be available to those in need (Table 1). This chapter will focus on the biological challenges for HIV-1 vaccine development and potential solutions. In addition, vaccine approaches that have completed or are undergoing efficacy testing, and new approaches being considered for advanced testing, will be described with a view toward the next steps needed to arrive at a preventive HIV-1 vaccine. Vaccine efficacy studies are performed in the context of other prevention modalities. To date, behavioral modification has been the only such intervention; however, circumcision has recently been shown to reduce HIV-1 infection and transmission rates. Active programs include education and risk-reduction counseling, female-initiated barrier methods, treatment of HSV-2 and other ulcer-causing diseases, and anti-retroviral (ARV) treatment of index partners. In addition, ongoing studies of pre-exposure ARV prophylaxis and microbicides will expand the alternative preventive measures over the next few years. An HIV-1 vaccine will need to provide increased efficacy compared with other prevention approaches and/or provide added value when combined with these approaches. Particularly when considered as an adjunct to other prevention measures, even a partially effective HIV vaccine could make a major contribution to controlling the spread of HIV. The current vaccine approaches being advanced into efficacy testing are based on the rationale that HIV-specific CD8þ T-cell responses elicited by vaccine prior to exposure to

HIV may rapidly control the spread of HIV and achieve a viral load set point lower than expected for a person infected without prior vaccination. Lower viral load would also make it much less likely that an infected vaccinee would transmit HIV to another person. However, because current vaccine antigens cannot induce a broadly neutralizing antibody response, vaccine-induced protection will rely on rapidly clearing newly transmitted virus. While it is possible this could result in abortive infection and a reduction in acquisition, it is plausible that current candidate vaccines might exert their effect through reduction of viral load, resulting in delayed disease progression and diminished secondary transmission. This type of ‘‘partially effective’’ vaccine, while potentially a valuable component in the effort to control epidemic HIV, will face unique challenges in both clinical and laboratory evaluation, and in the regulatory pathway to licensure (1).

Biological Challenges for Achieving VaccineInduced Immunity Despite more than two decades of intense biomedical research into the biology of HIV and the pathogenesis of HIV/AIDS, a vaccine for preventing infection has not been developed. Basic questions on the basis and mechanisms of vaccine-induced immunity remain, and immunological correlates of protection in humans remain unknown or controversial. Inherent biological properties of HIV pose challenging obstacles to vaccine development and include: (i) lack of natural immunity with no proven case of complete viral clearance and cure from HIV infection; (ii) evidence for superinfection; (iii) extreme genetic diversity that makes vaccine antigen selection difficult and increases likelihood of escape from immune responses (Fig. 1); (iv) structural features of the envelope that interfere with the induction of broadly neutralizing antibodies and limit their access to vulnerable structures when present; (v) predilection for infecting HIV-specific CD4þ T lymphocytes, providing a potential conduit rather than a barrier to infection; (vi) immune evasion including interference with antigen presentation, molecular latency, and persistence in reservoirs of long-lived CD4þ T cells, infection of immunoprivileged sites, such as the brain and eye that serve as a sanctuary from immune responses, and sequestration of virus in extracellular spaces in lymph nodes, where virus is not cleared by T cells or antibody; (vii) rapid destruction of memory CD4þ T lymphocytes in the intestinal tract, suggesting a potential role for mucosal

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Table 1 Ideal Characteristics of an HIV-1 Vaccine Safe and relevant for susceptible populations Safe for adults, prepubescent children, and infants Protection from intravenous and mucosal exposures Added value to other prevention approaches Effective against diverse forms and subtypes of virus Cell-associated and isolated virions Viruses with CCR5 and CXCR4 coreceptor usage Immune responses with specificity for antigens encoded by multiple subtypes Broad multiepitope response to limit immune escape Induce durable immunity to prevent infection or rapidly clear virus Reduce peak viremia after primary infection and diminish transmission to others Preserve CCR5þ memory CD4þ T cells Limit genetic variation to prevent immune escape Prevent integration and latency to prevent persistent infection Avoid establishment of infection in immunoprivileged sites Circumvent virus sanctuaries in lymph node extracellular spaces Accessible to developing countries Affordable Simple to administer Easy to distribute Stable in locations with marginal options for cold storage

immunity; and (viii) the limitations of animal models that may or may not accurately predict correlates of immunity or disease progression in humans.

Prospects for Achieving Vaccine-Induced Immunity Despite the expanding epidemic and many biological challenges imposed by HIV, there are reasons to believe that it is possible to generate vaccine-induced immunity. First, prospective analyses of HIV transmission in serodiscordant couples in Uganda indicate that heterosexual transmission is relatively inefficient (2). On average, several hundred exposures are required to cause one infection in settings of sexual transmission (3) or needlestick injuries (4). Therefore, vaccines that induce even modest improvement in antiviral defenses may have a profound impact on the transmissibility of HIV. Second, based on analysis of HIV isolates in acute infection, most individuals are infected with an inoculum of limited genotypic and phenotypic diversity (5,6). This feature improves the chances that even modest preexisting vaccine-induced immunity could prevent or modify infection. Third, data from lentivirus infection in nonhuman primates (NHPs) have

Figure 1 HIV-1 genotypic diversity. This map shows the global distribution of genotypically defined HIV-1 subtypes and recombinants. HIV-1 strains are currently organized by phylogenetic analysis of full-length genomes and divided into three major groups. Groups O and N are uncommon and found primarily in Cameroon. The M group contains nine subtypes and 16 CRFs. Each distinct subtype can have ~30% nucleotide mismatch in envelope and ~15% mismatch in gag from other subtypes, and CRFs have identifiable regions of intersubtypic recombination. Abbreviation: CRFs, circulating recombinant forms. Source: Courtesy of Francine E. McCutchan, U.S. Military HIV Research Program, Rockville, Maryland.

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Figure 2 Potential benefits of T-cell-mediated, vaccineinduced immunity. About three weeks after primary HIV-1 infection, a high magnitude viremia occurs that is associated with a massive depletion of CCR5þ memory CD4þ T cells. These early events are a major determinant of future disease progression in the infected individual. In addition, the peak viremic time period is thought to be when the majority of secondary transmission events occur. While CD8þ T-cell-mediated immunity may not prevent infection, animal models predict that it can reduce peak viremia, potentially delaying disease progression and reducing spread of infection to others (middle curve). If sufficiently rapid virus clearance can be achieved because of a high precursor frequency of vaccine-induced HIV-1-specific CD8þ T cells, it may be possible to prevent persistent infection (lowest curve). This question can only be answered in the context of an efficacy trial.

shown that vaccine-induced CD8þ T-cell responses correlate with control of viremia even when animals are challenged with highly pathogenic simian immunodeficiency virus (SIV) (7,8). Johnson and colleagues have shown that heterologous primeboost immunization using a DNA/MVA (deoxyribonucleic acid/modified vaccinia Ankara) combination achieves reduction in peak viral load (VL) and sustained reduction of the set point VL in NHPs infected by repeated vaginal challenges with SIV (9). Recent evidence suggests that a major factor in determining the magnitude of immunodeficiency is the degree of depletion of CCR5þ CD4þ memory T cells that occurs during the first few weeks of infection (10). Protection of these cells, largely found in the gastrointestinal tract, with preexisting vaccine-induced immunity slows disease progression in NHPs infected with SIV (11,12), suggesting that subtle influences on the timing, magnitude, and quality of the lentivirusspecific immune response may have a significant impact on the dynamics of disease progression (Fig. 2). In addition, there are examples of natural antiviral immunity from studies of highly exposed, uninfected commercial sex workers (13,14) and longterm nonprogressors (15,16). Host factors, such as specific immune-response genes can alter the clinical outcome of HIV-1 infection (17–19). These genes often affect the generation of CD8þ T cells, which are temporally associated with reduction in virus load (20,21) Using multiparameter flow cytometric analysis, a multifunctional T-cell response has also been associated with delayed disease progression (20). Long-term nonprogressors have a higher frequency of T cells producing multiple effector molecules after HIV-specific peptide stimulation than subjects with normal rates of disease progression. Collectively, these observations suggest that T-cell responses, particularly CD8þ T-cell responses against multiple specificities and effector functions, may confer a beneficial effect on HIV infection. Therefore, a major goal of current vaccine development efforts is to elicit broad, potent, and durable, HIV-specific T-cell responses to control the virus replication and reduce the likelihood of disease progression and transmission (Fig. 2). To contain naturally circulating viruses, vaccine must elicit protective immune responses to a broad spectrum of viruses or to highly conserved and functionally constrained antigenic domains of the virus. The genetic variation of HIV is daunting: HIV-1 evolves significant genetic adaptations to

escape ARV drugs or immune responses. The distinct epidemics around the world appear to be shaped by the regional collection of human leukocyte antigen (HLA) alleles and other host influences that result in the diversity of virus genotype (22). The diversity is amplified by the ability of coinfecting strains to recombine readily, often resulting in new circulating recombinant forms (CRFs) variants (Fig. 1). However, conserved regions within the structural proteins do not readily change because of the high cost to viral fitness, and those antigenic determinants provide a basis for achieving immunity against a broad variety of viruses. Genotypic diversity does not necessarily reflect antigenic diversity. Particularly for T-cell responses, many conserved epitopes allow crossclade recognition of diverse viruses. An innovative approach to antigen design involving in silico recombination of multiple sequences to maximize the number of potential T cell epitopes has been proposed as a way of broadening the response (23). These mosaic antigens will be tested in the next generation of HIV vaccine candidates. Most successful antiviral vaccines rely on neutralizing antibody for preventing infection (Fig. 3). Antibody responses tend to be more type-specific than T-cell responses, and there is currently not an antigen, either in natural infections or candidate vaccines that can consistently elicit broadly neutralizing antibodies. Passive transfer of antibodies can confer protection in NHP lentivirus infection (24), although at high concentrations and for short durations. These studies suggest that if a sufficiently high level of preexisting vaccine-induced neutralizing antibody was present, HIV-1 infection could potentially be prevented. It has been suggested that some transmitted viruses, particularly from clade C, may have slightly different gp160 glycosylation patterns and potentially a different neutralization sensitivity (25–27). The development of neutralizing antibody immunogens has benefited from an increased understanding of the structural basis of Env function and antibody-mediated neutralization. The atomic structure of binding sites for broadly neutralizing monoclonal antibodies against the CD4 binding domain of gp120 (28) and the membrane proximal region of gp41 (29) have been defined. Structureassisted design of HIV-1 envelope antigens provides a scientific path for achieving the goal of vaccine-induced neutralizing antibody (30).

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Figure 3 Immune response components important for different stages of virus life cycle. Most anti-viral vaccines with a known mechanism of protection rely on neutralizing antibody to prevent infection. Antibody is the only effector mechanism in the adaptive immune response that can attack an invading virus prior to infection of the first cell. Once cells become infected, cytolytic CD8þ T cells are the most important effector mechanism for viral clearance, although there may be a minor role for antibody-dependent cellular cytotoxicity or antibody-mediated complement-dependent cytolysis. If memory CD4þ T cells become quiescent and latently infected, or if virus is sequestered in the extracellular space in lymph nodes, or finds sanctuary in immunoprivileged sites like the brain or eye, there will be limited capacity for recognition or immune-mediated clearance.

ADVANCED CLINICAL EVALUATION OF CANDIDATE HIV VACCINES In the past 20 years, more than 50 distinct HIV/AIDS vaccine concepts have been evaluated in more than 30,000 volunteers globally. These studies include more than 120 small phase I trials that determine the initial safety and immunogenicity profile of a particular vaccine candidate. Selected approaches have prompted additional evaluation in 14 phase Ib trials to define dose and schedule better, and seven formal phase II studies have been designed to determine whether the safety and immunogenicity of the product qualified for efficacy testing. From these studies, three products have advanced to efficacy trials. The rationale and merits of these candidates are reviewed here. Phase III efficacy studies designed to support licensure of an early approach using monomeric HIV-1 envelope subunit proteins were completed and failed to show efficacy. An alternative approach using a poxvirus vector in combination with a subunit envelope protein boost completed phase III evaluation in July 2009. More recent efforts have specifically focused on eliciting T-cell-mediated immunity. One approach uses multiple doses of homologous replicationdefective adenovirus vector (rAd5) and another approach uses heterologous vector prime-boost using DNA combined with rAd5. The rAd5 alone was being tested in phase IIb, and the DNA prime/rAd5 boost approach is being advanced to a testof-concept efficacy trial.

Nonneutralizing Antibody Responses Elicited by Envelope Glycoprotein Antigens Initial approaches in the mid- to late 1980s to produce vaccine antigens focused on recombinant protein subunit products based on safety and available technology. Most products

were based on envelope glycoproteins, gp120, or gp160, because of their functional importance for virus attachment and entry, and as potential primary targets for neutralizing antibody. Recombinant subunit gp160 or gp120 has been produced in insect, yeast, or mammalian cells (31–35); however, Chinese hamster ovary (CHO)-derived recombinant gp120 (rgp120) emerged as the most immunogenic of the early subunit vaccines (33,35). Several properties of subunit proteins limited the utility of this approach. For instance, serum antibody titers induced by gp120 products have a short halflife (<6 months), and while they can be boosted, the titers generally achieve their peak level after the third or fourth injections. Repeated boosting does not significantly prolong the half-life. The rgp120 formulated in alum and produced by Genentech (South San Francisco, California, U.S.), the parent company of VaxGen, progressed to phase III efficacy evaluation in two trials. One placebo-controlled study in the United States, Puerto Rico, and the Netherlands utilized VaxGen B/B derived from HIV clade B strains LAI (one of the original T-cell lineadapted viruses) and GNE8 (a primary R5 isolate). This study enrolled 5108 gay and bisexual men and 309 women at high risk of HIV infection. The other placebo-controlled study utilized VaxGen B/E derived from HIV clade B LAI and clade E primary R5 isolate strain A244, and enrolled 4943 injection drug users in Bangkok, Thailand. The participants received vaccine at zero, one, and six months, then additional booster injections at six-month intervals. Based on rates of infection between placebo and vaccine recipients, there was no efficacy detected in either trial. The monomeric gp120 elicited antibody and CD4þ T-cell responses, but the antibodies were type-specific and failed to neutralize commonly transmitted primary R5 HIV-1 isolates (36).

Recombinant Poxvirus Vectors with or Without Recombinant Envelope Boosting The eradication of smallpox using replication-competent attenuated vaccinia was one of the greatest achievements of medical science. This legacy of vaccine efficacy and the development of technology to express recombinant genes from poxviruses (37) led to the development of recombinant poxviruses as potential vaccine candidates for other pathogens. Importantly, delivery of the vaccine by a vector allows endogenous production and processing of the antigen, thereby promoting major histocompatibility complex (MHC) class I epitope presentation and CD8þ T-cell induction. Poxvirus vectors that express Gag induce the production of pseudovirions from the infected cells (37–39), and may have some immunological advantages that can be optimized by altering vector construction (40,41). Early studies, beginning in the late 1980s, utilized live recombinant vaccinia (42,43), and they were indeed able to consistently induce long-lived CD8þ CTL responses in vaccinianaive subjects (44–46). At the same time, poxviruses encode gene products that inhibit immune responses to evade immune detection in vivo. In addition, the single recombinant gene expressed in these vectors must compete with many endogenous viral proteins for antigenic recognition. Additional concerns over the safety of replication-competent vaccinia, diminished immunogenicity caused by prior vacciniaseropositivity, and product supply issues have diminished enthusiasm for this vector and prompted a reevaluation of recombinant poxvirus strategies.

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Recombinant canarypox vectors emerged in the mid 1990s, and were evaluated in a series of clinical trials. Canarypox is grown and manufactured in chicken embryo fibroblasts (CEFs), but is replication incompetent in mammalian cells. Therefore, it can deliver its recombinant genes to the cytoplasm, where it provides all the machinery necessary for gene expression, but is unable to propagate itself to cause primary disease, and has been proven safe even in profoundly immunocompromised animals. A series of products were evaluated that expressed gp160 only (vCP125); gp120, gp41 (transmembrane), Gag, and Protease (vCP205); all the genes from vCP205 plus selected epitopes from Nef and Pol (vCP300); or all the genes from vCP300 plus E3L and K3L genes from vaccinia that inhibit dsRNA, interferon-inducible protein kinase, PKR, and inhibit apoptosis of the infected cells (vCP1452). The evaluation included: dose-ranging studies, a variety of injection schedules, combined administration by parenteral and mucosal routes, and combination with rgp120 envelope products. Canarypox vectors in general are well tolerated at doses up to 107 pfu, although local and systemic reactogenicity increased at the higher doses. HIV-specific antibody responses after recombinant canarypox immunization alone is weak, but subsequent boosting with purified recombinant envelope subunit protein induces HIV-specific antibody titers of the same or higher magnitude and quality as three or four inoculations of the purified recombinant envelope subunit protein alone (47,48). The sequence of recombinant canarypox priming followed by envelope subunit protein boosting results in a slightly longer antibody half-life than immunization with purified protein alone. HIV-specific CD8þ cytotoxic T lymphocytes (CTL) can be detected in fresh peripheral blood mononuclear cells (PBMCs) from subjects immunized with recombinant canarypox virus vectors, and in a subset of individuals the activity was detectable for more than two years. The activity detected in classical 51Cr release assays requires two weeks of in vitro stimulation and is detected in about 20% of subjects at any given time point (47–51). Although these responses may be of marginal strength, recombinant canarypox-induced CTL responses have been shown to lyse target cells infected with primary R5 HIV-1 isolates from multiple clades (52). In addition, CD8þ CTL effectors have been isolated from rectal mucosa (53). HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T-cell immunity in HIV-1-uninfected individuals, and both classical MHC class I–restricted cytolytic activity and nonlytic CD8þ-mediated HIV-1 suppression have been demonstrated in recipients of recombinant canarypox vaccines (54). The vCP1452 canarypox construct in combination with VaxGen rgp120 B/B was evaluated in phase II studies in the United States and did not achieve the predefined criteria to advance to efficacy evaluation (55). The vCP1521 construct with vCP205-like properties has been advanced into a 16,402 persons efficacy trial in Thailand. This canarypox vector expresses an envelope gene derived from a clade E HIV-1 isolate, and was given in combination with the VaxGen B/E rgp120 for efficacy evaluation in persons at risk from heterosexual HIV transmission. The initial modified intent-to-treat analysis, announced in September 2009, showed a point estimate of 31.2% reduction in acquisition and no change in viral load among those who became infected. This is the first evidence of HIV vaccine efficacy in humans and will facilitate the search for immune correlates of protection and guide the development of better animal models.

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Replication-Defective Viral Gene Delivery Vehicles Alone or in Combination with DNA Vaccination As the focus shifted away from monomeric envelope subunit vaccines in the mid 1990s, several new vector-based strategies for inducing strong CD8þ CTL responses emerged. Several gene delivery approaches of viral antigens have shown promise in NHPs, including DNA plasmids, replication-defective recombinant adenovirus vector (rAd), recombinant MVA, recombinant Venezuelan equine encephalitis virus (VEE), recombinant adeno-associated virus (AAV), recombinant vesicular stomatitis virus (VSV), recombinant poliovirus, replication-competent rAd, or combinations of DNA with one of the virus-based vectors (56). Of note, the induction of SIV-specific IFN-g ELISpot responses by priming with DNA followed by rAd5 boosting was shown to correlate with increased survival and delayed disease progression (8,57), reduction in viral load, and preservation of CCR5þ central memory CD4þ T cells (58) in NHPs. Recombinant Ad5 vectors have advanced to efficacy evaluation with or without prior priming with plasmid DNA expressing matching vaccine antigens. Though both stimulate cellular immunity with high frequency in humans, the qualitative and quantitative nature of the immune responses elicited by rAd alone versus DNA prime rAd boost is quite distinct, suggesting that each modality merits independent evaluation for efficacy. Delivery of naked DNA by plasmids is the simplest platform for introducing genes encoding vaccine antigens into target cells. A major advantage of vaccination with DNA is that no antivector immunity is induced. Another positive feature is that the only antigen produced is the specified vaccine antigen, so that there is no competition for antigen processing and presentation that may compromise more complex vector systems. While early attempts to immunize humans with plasmid DNA resulted in limited immunogenicity in clinical vaccine trials, recent studies of the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Vaccine Research Center (VRC) 4-plasmid and 6-plasmid products encoding envelope constructs from subtypes A, B, and C and Gag, Pol, and Nef from subtype B have shown consistent immunogenicity (59,60). A variety of technical improvements, including codon-modification, improved enhancer activity through the addition of the human T-cell lymphotropic virus (HTLV)-1 translation enhancing R region to the cytomegalovirus (CMV) promoter, and expression of each antigen from individual plasmids has improved expression levels as well as the potency and breadth of immunogenicity (59,61). Delivery of plasmid DNA by a needle-free injection device, Biojector1, may also be a factor in the improved immunogenicity relative to prior efforts at DNA immunization (unpublished observations). Replication-defective rAd5 vectors have been evaluated both alone and as a booster immunization in DNA-primed subjects. In NHPs, this prime boost combination stimulates a more potent immune response than a combination of DNA priming and rMVA boosting (62). Studies of prototype vaccines in the SHIV 89.6P challenge model have demonstrated the efficacy of combined DNA and rAd5 immunization in controlling subsequent viremia, and diminishing disease progression (63,64). rAd vectors are produced by inserting the recombinant genes of interest into the E1 gene cassette of the adenovirus genome, and producing the rAd particles in a producer cell line that is compliant with regulatory requirements and constitutively expresses complementary E1 genes (PER.C6 or 293-ORF6). In addition, portions of the E3 or E4 region can be

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removed from the vector genome to make room for larger recombinant genes and to diminish the production of adenovirus structural proteins and other proteins involved in immune avoidance. Both Merck & Co., Inc. and the VRC, NIAID at the NIH have performed clinical trials using rAd5 vectors. Merck first evaluated a clade B gag-expressing rAd5, and advanced a multivalent rAd5 vaccine, containing vectors expressing Gag, Pol, and Nef from clade B into efficacy testing. The Merck 502 study (STEP) conducted by the HIV Vaccine Trials Network (HVTN) began in December 2004, and enrolled 3000 subjects in clade B epidemic regions of the Western Hemisphere, Australia, and New Zealand. The HVTN Merck 503 study (Phambili) was conducted in South Africa, a predominant clade C epidemic, and enrolled 801 subjects beginning in February 2007. An interim analysis in September 2007 led to termination of the STEP and Phambili trials. Exploratory analyses of Ad5 antibody-positive men found a hazard ratio of vaccine to placebo recipient was 2.3 (95% CI 1.2– 4.3) and in uncircumcised men was 3.8 (95% CI 1.5–9.3), suggesting a trend toward increased risk of acquisition in vaccine recipients within these groups. In addition, early HIV-1 viral load was not reduced in vaccine recipients (65). Therefore, a vaccine that induced HIV-1 Gag- and Pol-specific CD8+ T cell responses in a majority of subjects did not reduce viral load and in a subset of individuals may have increased the risk for HIV-1 infection. The biological basis for this effect is unknown. The VRC rAd5 vector is a multivalent, multiclade product that expresses the gag and pol genes from clade B and modified envelope genes from clades A, B, and C, matching those in the DNA product described above. Phase I clinical trials have shown that the rAd5 is generally well tolerated, but at the 1011 PU dose, a short-lived syndrome of headache, myalgia, malaise, and fever of moderate severity sometimes occurs within 24 hours after vaccination. A milder version of this symptom complex is seen occasionally in recipients of the 1010 PU dose that is being evaluated in larger trials. The multiclade rAd5 alone induces T-cell responses in the large majority of subjects (66), and subjects primed with DNA T-cell and antibody responses following rAd5 boosting show several fold higher immunity than for either vaccine modality given alone. In addition, the T-cell responses elicited by the heterologous prime-boost combination of DNA followed by rAd5 induced a more polyfunctional T-cell response. This vaccine concept was evaluated in both phase I and II studies in the United States, Jamaica, Haiti, Brazil, Rwanda, Uganda, Kenya, Tanzania, and South Africa. A test-of-concept Phase IIb efficacy trial (HVTN 505) began in mid 2009. The study is expected to enroll 1350 Ad5-seronegative, circumcised men in North America. Preliminary endpoint analysis is anticipated in mid 2012. The major challenge facing rAd vectors, especially those intended for use in developing country settings, is the potential attenuating effect of preexisting adenovirus immunity. Current candidate vaccines are produced using an adenovirus serotype 5 (Ad5) packaging system, and Ad5 seroprevalence is high throughout the world. In developing countries 80% to 90% of adults are seropositive for Ad5, and about 50% have high titer anti-Ad5 activity (67). Preexisting Ad5 antibody was associated with a diminished magnitude of T-cell responses when rAd5 was used alone (66). Priming with DNA mitigates the attenuating effects of Ad5 antibody, particularly preserving HIV-1specific antibody responses, but persons with the highest Ad5 titers may still have lower T-cell responses even when primed

with DNA. To address this concern, a variety of alternative adenovirus serotypes and chimeric viruses have been designed as vectors and studied in animal models. Ad35 and Ad26 are relatively rare serotypes, and have both been shown to be immunogenic as vectors in NHPs. Another approach has been to modify the hexon protein to which much of the antiadenovirus neutralizing antibody is directed. By substituting the seven variable loops of the Ad5 hexon with those of the rare Ad48 serotype, Roberts et al. created an Ad5/Ad48 hexon loop chimera (HVR48) with the immunogenicity profile near that of rAd5, but resistant to Ad5 neutralizing antibody (68). These three alternative adenovirus serotype and chimeric vectors expressing HIV genes began clinical testing in 2008. In addition to the advanced evaluation of rAd vectors, new replication-defective poxvirus vectors (MVA and NYVAC) evaluated in combination with DNA-priming have shown promising immunogenicity in phase I clinical trials, and are being considered for phase II testing (69,70). The replicationdefective vaccinia vector elicits T-cell responses characterized by CD27-intermediate and CD45RO-negative surface phenotypes, as opposed to the CD27þCD45ROþ phenotype more typical of rAd-induced T-cell responses, and has a high degree of polyfunctionality. This indicates there are vector-dependent qualitative differences in vaccine-induced T-cell responses, and raises the possibility that these differences might influence the level of vaccine-induced protection (71).

SUMMARY OF CURRENT STATUS AND FUTURE PROSPECTS The aim of this chapter was to highlight major challenges for HIV vaccine development and to review the vaccine candidates and concepts advancing to efficacy evaluation. This discussion is by no means exhaustive, and interested readers are referred to other chapters contained in this volume on specific vaccine modalities and recent reviews that provide more detail (72,73). HIV poses unprecedented challenges for vaccine development that will take sustained, intense, and thoughtful efforts to overcome. While significant biological and logistic obstacles remain, vaccines remain the best hope for controlling the HIV/AIDS pandemic. The next critical steps are to 1) define correlates of vaccine-induced immune protection, 2) identify structures that elicit broadly neutralizing antibodies, and 3) develop next generation vaccines that elicit improved T cell and mucosal immunity.

ACKNOWLEDGMENTS We acknowledge Dr. Mary Lou Clements who wrote the first HIV vaccine chapter for this book. Many of the studies reported here were influenced by her work prior to her untimely death on September 2, 1998, caused by the SwissAir 111 accident. We thank Dr. Julie E. Ledgerwood who provided constructive criticism and Dr. Francine McCutchan for providing Figure 1.

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56 Vaccine Strategies to Prevent Dengue Robert Edelman Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Stephen S. Whitehead Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

David W. Vaughn GlaxoSmithKline Biologicals, King of Prussia, Pennsylvania, U.S.A.

BACKGROUND Dengue fever (DF) is an acute Flavivirus infection transmitted by several species of Aedes mosquitoes. Dengue virus (DENV) has four antigenically related serotypes, DENV-1, DENV-2, DENV-3, and DENV-4. Infection with any one of the four serotypes can produce a broad spectrum of clinical illness, including asymptomatic infection, mild febrile illness, classic DF, and dengue hemorrhagic fever (DHF) including dengue shock syndrome (DSS). DENV most often produces a self-limited, febrile illness, DF, which is characterized by fever, headache, eye pain, myalgia, arthralgia, and rash. In its most severe form, DENV infection can lead to DHF/DSS, which can rapidly progress to death. Fatality rates in patients with severe dengue vary from less than 1% to more than 30%, depending on diagnostic acumen and availability of intravenous fluids and blood for treatment of hypovolemic shock caused by plasma leakage and hemorrhage (1). These dangerous forms of the disease occur in an estimated 5% to 10% of dengue patients, and children are particularly at risk in countries with endemic circulation of multiple DENV serotypes. Patients with severe dengue require hospitalization, and 30% to 40% of such children progress to DSS. Lifelong immunity to the infecting DENV serotype occurs among those who recover from the infection. During the past 50 years, dengue has evolved into one of the world’s major infectious diseases (2). Over 2.5 billion people are at risk of dengue in over 100 countries, and Aedes aegypti and dengue epidemic activity are now widely distributed in the tropics and subtropics (Fig. 1). An estimated 50 to 100 million dengue infections and 500,000 DHF/DSS cases occur annually, which are concentrated in Southeast Asia, the Western Pacific, and Central and South America (2). The simultaneous or sequential co-circulation of two or more DENV serotypes in a locale is associated with outbreaks of severe DF and DHF/DSS. The majority of severe dengue cases in most endemic countries occur in children younger than 15 years. In most countries of Southeast Asia endemic for dengue, nearly all of the adult population has been infected with one or more DENV serotypes. In countries where DENV is newly introduced, the

illness occurs in individuals of all ages and in all socioeconomic sectors. Dengue is now a leading cause of morbidity in American and European travelers and military personnel, rivaling or exceeding malaria in many countries (4). Flavivirus vaccines have proved effective to reduce or prevent disease from yellow fever (YF), Japanese encephalitis (JE) and tick-borne encephalitis (TBE) viruses (5–7). However, a licensed dengue vaccine has remained elusive (8). Dengue has become impossible to eradicate and difficult to control because of large susceptible populations in the tropics, increased regional and international travel, failure to sustain A. aegypti control programs, and perhaps the global emergence of more virulent genotypes of DENV (9,10). Policy makers in Southeast Asian countries have agreed that a dengue vaccine is urgently needed (11). This urgency is shared by the U.S. military and the World Health Organization (WHO) (12). In addition to background information on DENV and DENV immunity, we will discuss different vaccine development strategies, including replicating and non-replicating dengue vaccine candidates (Table 1), potential uses of vaccines for the global control of dengue, unresolved vaccine and public health issues, and the Pediatric Dengue Vaccine Initiative. Since the previous chapter was published in 2004 (8), several live attenuated tetravalent dengue vaccine (TDV) candidates have transitioned into phase I and II clinical trials in the United States and Southeast Asia, and are poised for trial in the Caribbean and Central America. Phase III field trials of one or more TDVs are anticipated to begin before 2010 in Central and South America and Southeast Asia (29,30).

Dengue Viruses DENVs are 40- to 50-nm enveloped spherical virions with a core of single-strand positive-sense RNA complexed with nucleocapsid protein (1). Viral genomes are 11 kb in length and encode three structural proteins [capsid (C), membrane (prM, M), and envelope (E)] and seven nonstructural proteins (NS1, NS2a/b, NS3, NS4a/b, and NS5). The viruses

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Figure 1 World Distribution of Dengue, 2003. Source: Adopted from Ref. 3.

preferentially infect cells of the reticuloendothelial lineage, particularly macrophages and dendritic cells (31). These antigen-presenting cells may be important in dengue pathogenesis and essential for direction of primary immune responses. After binding to cellular receptors, viruses enter the cytoplasm where their RNA is released from the nucleocapsid, translated, and transcribed (32). Maturation of virions occurs in the cytoplasm, with assembly in the endoplasmic reticulum; progeny viruses bud into intracellular vesicles and are released from the cell surface (33). Both structural and nonstructural viral proteins are expressed on the surface of the infected cell. The four DENV serotypes are members of a single antigenic complex in the family Flaviviridae and share 60% to 80% protein sequence homology (34). Despite significant genetic diversity among virus strains, both among and within different serotypes, there is good congruence between traditional serological classification of DENV isolates and classification based on genetic relatedness. However specific genotypes within a serotype are associated with disease of greater or lesser severity (10). The mechanisms leading to increased viral virulence of certain genotypes are under investigation, but there is no evidence that increased mutation rates or recombination has created new variants or genotypes (10).

The adaptive immune response to DENV infection contributes to the resolution of infection and plays a critical role in protecting against reinfection. Since it is this same immune response that is likely to also play a role in the enhancement of disease severity seen in patients with DHF/DSS, immunization against dengue disease must address the issues of protective immunity and the proposed pathogenic role of immune responses in patients with DHF/DSS. The induction of protective levels of neutralizing antibodies is the major goal of immunization, since the presence of neutralizing antibodies directed against the E protein is generally associated with protection against DENV infection. Robust neutralizing antibody responses develop after DENV infection and are believed to provide lifelong protection against reinfection with the same DENV serotype and short-lived protection of only a few months duration against a heterologous DENV serotype (35–37). This short period of cross-protection has been associated with the presence of cross-reactive neutralizing antibodies, which wane rapidly after infection (38). Live attenuated virus (LAV) vaccines and nonliving vaccines, such as inactivated virus vaccines, virus-like particles, or DNA vaccines, each readily induce both neutralizing antibodies and protective immunity. Although the role of DENV-specific cellular immunity in protection against reinfection appears to be minor (39), T-cell-mediated immunity to DENV, by analogy to West Nile virus, is a significant contributor to viral clearance (40–42). Immune responses to DENV mediates not only protection from disease but also appears to be a major factor in the pathogenesis of DHF/DSS, although other factors such as virus strain virulence and host genetic factors may play a role (10, 43–45). The exact immunological mechanisms that mediate enhanced disease remain incompletely defined. However, a strong association of severe disease in humans undergoing a heterotypic secondary DENV infection has been established (46–50). This enhanced disease severity observed after secondary infection by a different DENV serotype is believed to be mediated primarily by preexisting, non-neutralizing, heterotypic antibodies that enhance access of DENV to FcgR-bearing cells. Such cells presumably would be inefficiently infected in the absence of antibody. This results in an increase in both the total number of FcgR-bearing cells infected and the total amount of virus produced. This increase in virus replication contributes to the increased titer of virus in the blood of

Table 1 A Partial list of Dengue Vaccine Candidates Under Development as of July 2007 Vaccine type

Vaccine developer

Live attenuated Live attenuated Live attenuated, chimeric

WRAIR/GSK Biologicals Mahidol University/Sanofi Pasteur NIAID/Butantan Foundation, Panacea Biotec Ltd., and Biological E. Ltd. Acambis/sanofi Pasteur CDC/InViragen WRAIR Hawaii Biotech U.S. Navy Medical Research Center Oswaldo Cruz Foundation

Live attenuated, chimeric Live attenuated, chimeric Inactivated virus Subunit DNA

Serotype, clinical testing status

References

Tetravalent, phase II Tetravalent, discontinued Monovalent (1–4), phase II

Edelman (13), Sun (14,15) Kitchener (16), Kanesa-Thasan (17) Blaney (18), Durbin (19–21)

Tetravalent, phase I Preclinical Phase I Preclinical Phase I Preclinical

Guirakoo (22,23) Huang (24) Putnak (25,26) Putnak (26) Blair (27) Costa (28)

Abbreviation: WRAIR, Walter Reed Army Institute of Research; GSK, GlaxoSmithKline Biologicals; NIAID, National Institute of Allergy and Infectious Diseases, National Institutes of Health; CDC, Centers for Disease Control and Prevention.

Chapter 56: Vaccine Strategies to Prevent Dengue

DHF/DSS patients (48,51). This phenomenon is termed antibody-dependent enhancement (ADE) (52,53). Immune activation and extensive tissue injury caused by augmented virus replication, complement activation, and apoptosis presumably mediate the pathologic events of DHF/DSS (54,55). Both the effective role of antibody in controlling DENV disease and the detrimental role of antibody in enhancing disease can be observed during the first year of life. In endemic areas, dengue in infants less than four months is unusual and indicates that passively transferred maternal antibodies can protect the infant in the absence of fully developed cell-mediated immunity (56–58). Conversely, a role for ADE in the development of DHF/DSS is suggested by the timing of DHF/DSS that occurs in infants, generally between the ages of 6 and 12 months in endemic areas (56,57). When the maternally derived antibody titer to DENV declines below a protective level by approximately six months, infants are actually at an increased risk for the development of DHF/ DSS for a short window of time despite the fact that they have never been infected with a DENV and lack DENV-specific cellular immunity. After the complete degradation of maternally derived antibodies, infants lose the enhanced susceptibilit y t o D HF/D S S. ADE h as al so bee n de mon stra ted experimentally by passive transfer of monoclonal antibodies in rhesus monkeys (59). These observations strongly suggest that preexisting antibodies in the absence of DENV-specific cellular immunity are sufficient to promote the increased virus replication seen in DHF/DSS mediated in part by ADE (55,60,61). Although this feature of DENV infection requires careful consideration during implementation of a vaccination program against DENV, existing evidence indicates that a vaccine that induces sustained neutralizing antibody responses against each DENV serotype will be effective at preventing disease and will not contribute to enhanced disease.

A BRIEF HISTORY OF DENGUE VACCINE DEVELOPMENT Blanc and Caminopetros were the first to publish an approach to dengue vaccine development in 1929 by attenuating DENV in blood with ox-bile (62). At that same time, Simmons, St. John, and Reynolds developed the U.S. military’s first inactivated dengue vaccine by grinding DENV infected A. Aegypti mosquitoes in a salt solution and chemically pure phenol and formalin (63). While these injections failed to prevent subsequent infection and disease, some of the volunteers developed only mild cases of dengue. The first successful dengue vaccine was reported in 1952 by Sabin and Schlesinger, who attenuated the ‘‘Hawaiian’’ strain of DENV-1 in mouse brain by serial passage, and then used this mouse brain vaccine to protect 16 volunteers against the bites of infected A. aegypti mosquitoes (35). The modern era of DENV propagation in tissue culture began in 1971, which promised a safer vaccine substrate (64). Recombinant DNA technology has catalyzed more recent advances in vaccine development (12).

DIFFERENT DENGUE VACCINE DEVELOPMENT STRATEGIES Rationale for Tetravalent Dengue Vaccines The current strategy to develop a combined vaccine against all four dengue serotypes is supported by three, key epidemiological and immunological facts. First, a primary infection with

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one serotype may induce long-term protective immunity to reinfection that persists for years against the homologous serotype but immunity lasts for only several months against heterologous serotypes (35). Thus, no single serotype can provide long-term protection against the other three serotypes. Second, one or several serotypes can circulate simultaneously in any locale, and serotypes can change unpredictably from one season to the next. Thus, one cannot reliably predict if a monovalent vaccine would protect during the first dengue season and in subsequent seasons in any locale. A tetravalent vaccine will better protect travelers and troops rapidly deployed to tropical areas where several DENV serotypes cocirculate over time. Third, as described in the section ‘‘Immunity to Dengue,’’ dengue differs from other hemorrhagic fever infections in that DENV infections are more severe in individuals who have acquired dengue antibodies actively from a previous DENV infection, or passively from mothers before birth (36,50). Thus, DHF/DSS cases are associated with prior infection and are apparently mediated by non-neutralizing antibodies that are residual from an earlier DENV infection. ADE has provided an explanatory hypothesis whereby preexisting, cross-reactive DENV antibodies facilitate DENV entry into target cells, thereby increasing virus burden (52,54). A massive cellular release of virus and soluble DENV protein triggers complement activation, which together with inflammatory cytokines, synergize locally to trigger the vascular leak observed in severe dengue (55). The secondary infection hypothesis and ADE suggests that dengue vaccines must induce protective neutralizing antibodies to all four serotypes simultaneously rather than sequentially to avoid enhancement of dengue illness after subsequent infection.

Rationale for Live Attenuated Virus Dengue Vaccines Immune control of dengue may require a LAV rather than an inactivated (non-replicating) whole virus, recombinant subunit, or recombinant DNA vaccine for several reasons (65,66) First, control of epidemics requires rapid immunization with a single inoculation, or two closely spaced inoculations. Second, the vaccine should induce long-lasting neutralizing antibody levels mimicking that of natural DENV infection. Antibody titers should not wane to non-protective levels that may leave individuals susceptible to the immunopathological events associated with DHF/DSS if they are subsequently infected by a heterotypic DENV serotype. Third, low-cost efficient vaccines are needed to protect at-risk children in the tropics. By contrast to attenuated vaccines, inactivated and subunit vaccines normally elicit short-term immunity and require multiple booster inoculations to provide long-term immunity. They may fail to induce robust MHC class 1-restricted T-cell immunity that contributes to full protection against DENV infection. Thus, vaccine adjuvants would be required, which could be both expensive and potentially reactogenic. Because of the need for antigen-adjuvant formulations and because DENV does not grow to high titer in tissue culture cells, these vaccines are likely to be more expensive to manufacture than LAV. Their introduction could increase the cost of immunization, making them unsuitable for programs in developing countries (11). For these reasons, non-replicating dengue vaccines will remain a second choice for clinical development, or at least until the risk of LAV outweighs their benefit (65).

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Industry and government support will be essential for the prolonged and expensive field trials that lead to licensure. The leading live attenuated TDV candidates currently in clinical trial are discussed in the following text. Vaccine Candidates Passaged in Primary Dog Kidney Tissue Culture Researchers from Mahidol University (Bangkok, Thailand) and the Walter Reed Army Institute of Research (WRAIR, Silver Spring, Maryland) have used conventional methods to develop DENV vaccine candidates by passage in tissue culture cells. The Mahidol vaccine virus candidates were derived from clinical isolates grown first in either primary dog kidney (PDK) cells or primary African green monkey kidney (PGMK) cells and subsequently in fetal rhesus lung (FRhL) cells. Similarly, the WRAIR candidates were all grown in PDK cells with terminal passages in FRhL cells. Mahidol University vaccine candidates. The Mahidol University monovalent vaccines were reported to be well tolerated in flavivirus-naı¨ve adult and pediatric volunteers in Thailand (67–70) and in adults in the United States (17,71). Clinical evaluation identified passage levels for the monovalent candidates that induced minimal reactogenicity and a high level of seroconversion following one or two doses (72). However, when a tetravalent formulation was administered as a single dose, the predominant virus in the blood was DENV-3 and the neutralizing antibody response was directed largely against DENV-3 (17). Attempts to overcome this apparent viral interference by testing tetravalent formulations with altered levels of each virus component, particularly lower levels of DENV-3 and multiple doses, yielded mixed results with unacceptable levels of reactogenicity (16,69). Unfortunately, replacement of the DENV-3 component with a biologically cloned derivative was also highly reactogenic (73). Since the Mahidol vaccine candidates have not achieved a balanced immune response to each of the four components and systemic symptoms have occurred in recipients of the tetravalent vaccine, this vaccine has been discontinued by its industrial sponsor, Sanofi Pasteur. It will not be discussed further.

Walter Reed Army Institute of Research vaccine candidates. Researchers at WRAIR and the University of Maryland have used a series of studies in rhesus macaques and phase I clinical trials to identify appropriate passage levels for each of their PDK-derived vaccine candidates, based on the broad assumption that the virus becomes more attenuated with an increased number of passages (64,74,75). Following a single administration of the monovalent candidate vaccines to seronegative adult volunteers, the seroconversion rates ranged from 46% to 100% for each DENV serotype, with the highest rate achieved by DENV-1. However, while the DENV-2, DENV-3, and DENV-4 vaccines were only mildly reactogenic, the DENV-1 candidate was associated with increased reactogenicity with 40% developing fever and generalized rash (14). The under-attenuated nature of the DENV-1 component was also evident in early testing of the tetravalent formulation, that also indicated that the DENV-4 component was slightly overattenuated (13,14). To address these issues, the dosage levels of each component were adjusted and eventually, the DENV-1 component was replaced with a further passaged virus (PDK27 rather than PDK-20). The DENV-4 component was replaced with a lower passaged virus (PDK-6 rather than PDK-20) to improve immunogenicity. Current tetravalent formulations are in phase II testing in North America and Southeast Asia, sponsored by WRAIR and GlaxoSmithKline Biologicals.

As described in the preceding text, passage of DENV in PDK cells has lead to the accumulation of attenuating mutations. Unfortunately, with the exception of the Mahidol University DENV-2 vaccine component (76), the mutations contributing to the attenuation phenotypes of these vaccine candidates have not been identified. Because the vaccine candidates were never biologically cloned, genetic analysis or rederivation of the vaccine candidates has proven difficult (73). Genetically Engineered Vaccine Candidates In a separate vaccine strategy developed at the Laboratory of Infectious Diseases (LID), National Institute of Allergy and Infectious Diseases (NIAID, Bethesda, Maryland), the DENV4 full-length cDNA clone was used to engineer deletion mutations in the 30 untranslated terminal region (UTR) that conferred varying levels of attenuation in rhesus monkeys compared with the wild-type parent virus (77). Among the deletions created, which ranged from 30 to 262 nucleotides, the 30 172 to 143 deletion mutation, later referred to as D30, showed a desirable balance between level of attenuation and immunogenicity in monkeys. The DENV-4 virus containing the D30 mutation was subsequently evaluated in adult human volunteers and was shown to be safe, asymptomatic, and immunogenic at all doses administered (101–105 pfu/mL) (19,78). Because the structure of the DENV 30 UTR is well conserved among all four serotypes, it was reasoned that deletion of nucleotides analogous to the D30 mutation in each serotype would likely result in attenuation. Introduction of the D30 mutation into DENV-1 resulted in a vaccine candidate attenuated to levels similar to that observed in monkeys for DEN4D30 (79) that were well tolerated and highly immunogenic in humans at a dose of 103 pfu/mL (20). However, introduction of the D30 mutation into DENV-2 or DENV-3 conferred only weak or no attenuation, respectively (80,81). Because the DEN2D30 and DEN3D30 viruses are not suitable vaccine candidates, an alternative chimeric strategy based on the DEN4D30 vaccine candidate was used to create vaccine candidates for DENV-2 and DENV-3 (82). Other strategies to identify attenuating mutations for DENV have included the generation of point mutations throughout the virus genome. Chemical mutagenesis or paired charge-to-alanine mutagenesis has been successfully used to identify point mutations exhibiting a range of useful phenotypes, including temperature sensitivity, small plaque size, enhanced replication in Vero cells, reduced replication in mouse brain, reduced replication in severe combined immune deficiency (SCID) mice transplanted with human liver cells, and reduced infectivity for mosquitoes (83–85). Vaccine candidates bearing several of these mutations have been successfully tested in monkeys (86) and are currently being evaluated in humans. Substitution or deletion of noncontiguous nucleotides in the 30 -terminal stem-loop structure of DENV genome has yielded viruses DEN2mutF, with reduced replication in tissue culture mosquito cells (87), and DEN1mutF, with reduced replication in mosquito cells and attenuated replication in monkeys (88). It is possible that the mutF set of mutations could be introduced into the remaining DENV serotypes to develop a tetravalent vaccine in a manner analogous to that proposed for the D30 mutation. Chimeric Vaccine Candidates It has been possible to produce chimeric DENV in which the structural protein coding region of a flavivirus is replaced by

Chapter 56: Vaccine Strategies to Prevent Dengue

that from a specific DENV serotype, and this finding has been used to develop LAV vaccines (89). The goal of this approach is to use chimerization to bring together the immunogenic structural genes of a DENV and the attenuated nonstructural genes of an attenuated flavivirus to create safe vaccine candidates for each of the four DENV serotypes. For the generation of such chimeric viruses, it is generally accepted that the genetic background (or platform) should be attenuated. However, experience has shown that chimerization itself can lead to attenuation. Intertypic chimeric viruses created with a wildtype DENV-4 background and wild-type DENV-2 structural genes were significantly attenuated compared with either wildtype parent virus in mice, mosquitoes, or rhesus monkeys (90). Nevertheless, the use of an attenuated background further augmented the level of attenuation of the DENV-4/DENV-2 chimeric viruses (90). Several attenuated vaccine viruses or vaccine candidate viruses have been used as the platform for creating chimeric dengue vaccine candidates. These include the use of YF virus vaccine strain 17D (22,91–93), PDK passaged DENV-2 vaccine candidate PDK-53 (24), and genetically modified vaccine candidate DEN4D30 (80,90). Several promising chimeric dengue vaccine candidates have been successfully evaluated in preclinical studies and in human trials. In general, the chimeric vaccine candidates that have been evaluated in non-human primates have demonstrated decreased levels of viremia and a reduced number of viremic days compared to parental viruses from which the structural genes were derived (81,90,94–96). On the basis of the success of these non-human primate studies, selected vaccine candidates have recently been evaluated in human clinical trials. Acambis ChimeriVax vaccines. The ChimeriVaxTM (Cambridge, Massachusetts, U.S.) platform, based on YFV 17D and developed by Acambis, Inc. and Sanofi Pasteur, has been used to create chimeric vaccine candidates for each of the DENV serotypes. The monovalent ChimeriVax-DEN2 vaccine candidate was successfully evaluated in humans and shown to be safe and immunogenic (97). Reports of the phase I testing of the ChimeriVax tetravalent vaccine indicate that it is safe without any serious adverse side effects (Jean Lang, personal communication). For a more thorough discussion of the TM ChimeriVax platform and its use in creating flavivirus vaccine candidates, see chapter 52. LID/NIAID D30 vaccines. The LID, NIAID chimeric vaccine candidates DEN2/4D30 and DEN3/4D30 were generated and contain the prM and E of DENV-2 or DENV-3 on a DEN4D30 genetic background. These antigenic chimeric viruses have been shown to be highly attenuated for monkeys and it has been demonstrated that the observed attenuation was a result of chimerization as well as the presence of the D30 mutation (81,90). In addition, both the DEN2/4D30 and DEN3/4D30 vaccine candidates have very low oral infectivity for A. aegypti mosquitoes (81,90). Phase I testing of DEN2/4D30 has shown the vaccine candidate to be safe and immunogenic at a dose of 103 pfu (20), and clinical testing of DEN3/4D30 is currently underway. The DEN2/4D30 and DEN3/4D30 chimeric vaccine candidates have been combined with DEN1D30 and DEN4D30 to create a tetravalent formulation that has been shown to be attenuated (peak titers of <102 pfu/mL), broadly immunogenic, and protective in rhesus monkeys (98). The suitability of this tetravalent formulation for humans has not yet been studied, but is under consideration pending the outcome of the DEN3/4D30 clinical trial.

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Centers for Disease ControlDENV-2 PDK-53 vaccine. Using the attenuated DENV-2 PDK-53 vaccine strain developed by Mahidol University, researchers at the Center for Disease Control and Prevention, (CDC, Fort Collins, Colorado), have developed a set of chimeric vaccine candidates based on the three attenuating mutations of the DENV-2 PDK-53 strain, which lay outside the structural genes. The chimeric vaccine candidates have been shown to be immunogenic and protective in mice (24), and a tetravalent study in monkeys has been recently completed. Preclinical study of the vaccine candidates is ongoing and a phase I study in humans is anticipated. Other Types of Vaccine Candidates Inactivated vaccines have at least two advantages over live attenuated vaccines; research is not required to identify stably attenuated parent strains since wild-type strains can be used effectively, and the components in a multivalent inactivated virus vaccine may be less likely to interfere with one another. Both cell-mediated and humoral immune responses can be induced with an inactivated flavivirus vaccine as has been shown for JEV vaccine (99). Nonetheless, the use of an inactivated whole virus vaccine is accompanied by real and theoretical difficulties, most of which have been discussed previously under the section ‘‘Rationale for Live Attenuated Virus Dengue Vaccines.’’ These difficulties make inactivated DENV vaccines less attractive candidates for endemic areas, but they may be useful as a traveler’s vaccine or as part of a prime-boost strategy with live or replicating vaccines. Purified inactivated vaccines. A purified, inactivated DENV-2 vaccine has been manufactured by WRAIR and a DENV-1 equivalent will soon enter clinical trials (25,26,100). For preparation of the inactivated vaccine candidates, the viruses were propagated in qualified Vero cells and concentrated by ultrafiltration and purified on sucrose gradients. The high-titer purified virus (*109 pfu/mL) was then inactivated with formalin. The DENV-2 vaccine formulated with alum and other adjuvants induced high neutralizing antibody levels and protection against viremia in a primate model (26). Research directed toward optimizing virus yield from tissue culture would help to decrease the cost of manufacture. Subunit vaccines. Dengue antigens, primarily E proteins, have been produced in a number of expression systems to generate subunit vaccine candidates that have elicited moderate to high levels of antibody following immunization of mice. Although such vaccines are anticipated to be safe, it is likely that they will share some or all of the difficulties discussed previously concerning inactivated vaccines. To date, a DENV subunit vaccine has not been tested in humans. However, two studies in rhesus monkeys have recently been completed using monovalent DENV-2 or DENV-4 truncated E proteins. Guzman et al. immunized monkeys with four doses of 100 mg of E protein (DENV-4), using alum as an adjuvant and achieved partial protection against wild-type DENV-4 challenge (101). In collaboration with Hawaii Biotech, Inc., Putnak et al. immunized monkeys with two doses of DENV-2 E protein produced in drosophila cells and formulated with each of five different adjuvant combinations (26). The DENV neutralizing antibody titers prior to challenge varied widely, and the groups of monkeys receiving the highest dose of antigen along with two adjuvants were protected against viremia after challenge with wild-type DENV-2. Hawaii Biotech is currently manufacturing affinity-purified E protein for each of the four DENV serotypes and will soon initiate a phase I clinical trial.

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Recombinant vectored vaccines, including DNA vaccines. Although numerous recombinant vector systems have been used to express DENV antigens, the resulting vaccine candidates have achieved only limited success using modified vaccinia or adenovirus vectors and additional development of suitable vector platforms will be necessary (102,103). Rather than using live vectors to express DENV antigens, such antigens can be expressed from DNA constructs that are introduced into cells and subsequently translated into dengue antigens or subviral particles. DNA vaccines afford advantages over conventional vaccines including ease of production, stability and transport at room temperature, and decreased likelihood of replication interference. Preclinical evaluation of DNA vaccine candidates expressing the prM and E genes has been conducted by the Naval Medical Research Center (NMRC, Bethesda, Maryland). In recent experiments using genes from DENV-1 virus, it was shown that three doses of vaccine protected Aotus monkeys following challenge with wild-type virus (104). Partial protection against viremia after challenge of Aotus monkeys was also induced by a DENV-3 DNA vaccine (27). A clinical trial with an improved DENV-1 DNA construct delivered in three doses is currently underway. Nevertheless, recent experience with DNA vaccines for DENV has highlighted the necessity for multiple doses, experimental adjuvants and immunostimulatory motifs, and specialized injection equipment. Prime-boost strategies using a combination of live vectors or DNA vectors expressing the E protein have been investigated, but such strategies for TDV development seem too complicated for economical delivery and use in endemic areas.

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POTENTIAL USES OF VACCINES FOR THE GLOBAL CONTROL OF DENGUE The way vaccines might be used for dengue control in different epidemiological settings remains speculative, because we do not know the characteristics of vaccines likely to become licensed and their immunogenicity and duration of protection. The following uses and situations are at least plausible (29). The list is not intended to be exhaustive.

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The inclusion of a dengue vaccine in the national immunization program (NIP) of countries where significant DENV transmission occurs and the disease-burden is well established and likely to continue into the foreseeable future. Examples include much of tropical Asia, the western Pacific, Central and South America, and the Caribbean. DENV transmission exists in Africa, but severe dengue is not recognized there, and dengue vaccination will unlikely be implemented in Africa as a high priority. The inclusion of a dengue vaccine in a NIP raises the issues of possible interference between the dengue vaccine and the other NIP vaccines, and the optimal timing of dengue vaccination and the possibility of adapting it to the existing NIP schedule (105). Sustainability of dengue vaccination could be a major problem. ‘‘Catch-up’’ dengue vaccine campaigns in dengueendemic countries to immunize susceptible populations who have completed their NIP vaccinations, such as children through school age, adolescents, or adults. The actual age groups targeted would need to be identified through epidemiological studies. These older populations may be partially flavivirus-immune so that vaccine immunogenicity and safety needs to be assured.

In certain countries, the safety and efficacy of dengue vaccines in immunocompromised persons (including HIV-positive children) and pregnant women would need to be evaluated. The risk of infection with LAV dengue vaccines in such persons is not known. There are few reports of adverse effects of DENV infection on maternal and fetal outcomes, although any fever is a threat to pregnant women. It is also possible that immunocompromised persons are actually at decreased risk for severe dengue, because DHF/DSS may be triggered by accentuated immune responses to the infection. Nevertheless, it is prudent currently to delay use of LAV vaccines in these populations until proven safe. Mass vaccination as part of a time-limited campaign designed to eliminate dengue and DENV transmission in a defined location. Elimination of virus transmission and resultant herd immunity would likely be temporary unless sustained vaccination could be combined with simultaneous intensive mosquito control. Mass vaccination as part of the short-term management of a dengue epidemic. A short immunization schedule (e.g., a single injection) would be desirable. However, it may be difficult to ensure vaccines are administered safely in an emergency mass campaign. The clinical consequences of vaccinating asymptomatic individuals recently infected with DENV during the epidemic are unknown. Protection of nonimmune, temporary visitors to endemic areas (e.g., travelers, seasonal laborers, military personnel). Demand for vaccine is likely to be high in the absence of viral chemoprophylaxis. The optimal vaccine should rapidly induce high-grade protection. In contrast to vaccine applications discussed earlier, a relatively short period of protection may be acceptable, and for some visitors (e.g., wealthy travelers), a relatively high cost may be acceptable. Vaccine scheduling may be complicated by the need to vaccinate travelers with more than one flavivirus vaccine, for example, dengue and JE or YF vaccines, so that viral interference would need to be excluded.

SOME UNRESOLVED VACCINE AND PUBLIC HEALTH QUESTIONS A sampling of other unique and complex issues that surround attenuated TDV field trials (aside from the theoretical risk of vaccine-associated DHF/DSS) are summarized below.

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Can a TDV consistently achieve acceptable reactogenicity, 80% protective efficacy to all four serotypes in all countries at risk for DENV transmission, for at least three to five years (11)? Is this consensus vaccine response appropriate in all populations and clinical settings? Phylogenetic and epidemiological analysis suggest that more virulent genotypes are displacing those that have lower epidemiological impact (9). Fortunately, there have been no genetic changes creating new serotypes or antigenic variants of the four existing serotypes, so that one may expect the current TDV components to protect against all genotypes, whether virulent or not. Because there are no acceptable animal models, field trials must be designed to elucidate DV evolution and pathogenesis in addition to vaccine safety and efficacy.

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The relative protection afforded by neutralizing antibody versus cellular immunity should be clarified in future field trials (106). In the absence of antibody, can unequivocal evidence of immune priming provide a reliable correlate of protection? We need to develop in vitro tests capable of distinguishing protective from non-protective vaccine responses (106). WHO should endorse a standardized DENV antibody micro-neutralization assay. Several quantitative PCR assays close to being validated will permit detection of each of the four DENV serotypes in blood of trial participants. In what epidemiological settings can herd immunity, provided by partial vaccination of a population, decrease DENV transmission among unvaccinated persons? To what extent will herd immunity confound the analysis of vaccine protection? Will TDVs be safe and immunogenic in persons preimmune to other Flaviviruses, such as West Nile virus, JE virus, and YF virus? We are reasonably reassured that severe reactions would not occur in YF-immune persons (23,64). Safety needs to be confirmed in other Flaviviruses-immune individuals. Would TDVs be safe and immunogenic in HIV-infected and other immunosuppressed persons? LAVs are generally contraindicated in such individuals but attenuated TDV candidates will need to be tested eventually. The ethics of such studies may be questioned, but they can be addressed by a consensus of vaccine stakeholders and review boards. How do vaccine responses in infants and children differ from adults? Infants often respond to wild DENV infection with few symptoms, and preadolescent children are less incapacitated by DENV infection than adults. In fact, clinical attenuation as a function of decreasing age was noted in the first PDK-attenuated tetravalent vaccine trial in seronegative children (70). The immune and clinical responses to the vaccine need to be stratified by age in future field trials, to include Flavivirus-negative and positive participants. A consensus of investigators, vaccine developers, and national regulatory authorities are of the opinion that the short-term objective of phase III TDV field trials should be to protect against virally confirmed dengue disease of any serotype and of any severity, from mild dengue to DHF. The long-range objective would be to confirm efficacy against all four serotypes. To do this, geographically diverse field sites must be developed to insure that the vaccine can be tested against the four circulating serotypes and against the full range of clinical dengue infections.

PEDIATRIC DENGUE VACCINE INITIATIVE (PDVI) For the first time in 60 years an opportunity exists to put newly developed dengue vaccines into the field quickly (30). The Bill and Melinda Gates Foundation funded the PDVI in July 2003 with 55 million dollars for five years. The PDVI has been collaborating with vaccine researchers and manufacturers, governments, and the WHO to fund a program to accelerate the development and field-testing of dengue vaccines. More specifically, the PDVI program aims to (i) better define the

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global burden of dengue illness, and to elucidate the social and economic costs of dengue, particularly in countries that may introduce dengue vaccines; (ii) create a consortium of vaccine evaluation field sites in dengue-endemic countries; (iii) work with WHO to revise guidelines for assessing the safety, efficacy, and effectiveness of dengue vaccines used in large-scale clinical trials (phase IIb, III, IV); (iv) develop provisional dengue vaccination strategies; (v) develop, evaluate, and standardize diagnostic tests for DENV infection, for immunity to DENV, and for identification of persons at risk of immune enhancement; (vi) work with partners to develop and promote plans for national and international vaccine procurement and distribution; (vii) educate public and private organizations about dengue as a vaccine-preventable disease.

CONCLUSIONS Following successes with live attenuated 17D YF vaccine, formalin-inactivated and cell culture-derived JE vaccines, and formalin-inactivated TBE vaccine, decades of efforts to develop live attenuated dengue vaccines may be nearing a conclusion as the pace of dengue vaccine development is accelerating at an unprecedented rate. This is due in large part to the combination of technical advances (recombinant and reverse genetics and the availability of PDK and Vero cells for manufacture) and the availability of adequate funds for clinical field trials (provided by the participation of two, multinational, pharmaceutical companies and the Pediatric Dengue Vaccine initiative). As a result, two empirically derived, attenuated TDV have been in phase I and phase II clinical testing in the United States and Southeast Asia and one is poised for trials in Latin America. Several chimeric vaccines in early clinical trial in the United States provide additional, promising, vaccine options. Phase III field trials of one or more TDVs are anticipated to begin before 2010 in Central and South America and Southeast Asia. To support field trials, the WHO and PDVI are developing field study sites, standardizing viremia and antibody assays, and supporting basic and clinical research designed to elucidate protective responses and DHF immunopathogenesis. The advent of novel molecular techniques, such as expression of viral subunit proteins and construction of infectious clones, has led to dozens of candidate vaccines now in preclinical testing. If TDVs are proven protective and safe in the short and long term, the future of licensed dengue vaccines seems assured.

ACKNOWLEDGMENTS The authorship of this manuscript was supported in part by the Intramural Research Program of the NIH, National Institutes of Allergy and Infectious Diseases.

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24. Huang CY, Butrapet S, Tsuchiya KR, et al. Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development. J Virol 2003; 77(21):11436–11447. 25. Putnak R, Barvir DA, Burrous JM, et al. Development of a purified, inactivated, dengue-2 virus vaccine prototype in Vero cells: immunogenicity and protection in mice and rhesus monkeys. J Infect Dis 1996; 174(6):1176–1184. 26. Putnak JR, Coller BA, Voss G, et al. An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model. Vaccine 2005; 23(35):4442–4452. 27. Blair PJ, Kochel TJ, Raviprakash K, et al. Evaluation of immunity and protective efficacy of a dengue-3 pre-membrane and envelope DNA vaccine in Aotus nancymae monkeys. Vaccine 2006; 24(9):1427–1432. 28. Costa SM, Paes MV, Barreto DF, et al. Protection against dengue type 2 virus induced in mice immunized with a DNA plasmid encoding the non-structural 1 (NS1) gene fused to the tissue plasminogen activator signal sequence. Vaccine 2006; 24(2):195–205. 29. World Health Organization (WHO). Guidelines for the clinical evaluation of dengue vaccines in endemic areas. WHO Initiative for Vaccine Research (IVR). WHO/IVB/08.12, 1–28. 2008. Geneva, Switzerland: WHO. Available at: www.who.int/vaccinesdocuments. 30. Pediatric Dengue Vaccine Initiative. Strategic Plan. Dengue Vaccines: The Role of the Pediatric Dengue Vaccine Initiative. International Vaccine Institute, 2006:1–44. Available at: http:// www.pdvi.org/StrategicPlan.pdf. 31. Wu SJ, Grouard-Vogel G, Sun W, et al. Human skin Langerhans cells are targets of dengue virus infection. Nat Med 2000; 6(7):816–820. 32. Putnak JR, Kanesa-Thasan N, Innis BL. A putative cellular receptor for dengue viruses. Nat Med 1997; 3(8):828–829. 33. Hase T, Summers PL, Eckels KH, et al. Morphogenesis of flaviviruses. Subcell Biochem 1989; 15:275–305. 34. Kuno G, Chang GJ, Tsuchiya KR, et al. Phylogeny of the genus Flavivirus. J Virol 1998; 72(1):73–83. 35. Sabin AB. Research on dengue in World War II. Am J Trop Med Hyg 1952; 1:30–50. 36. Guzman MG, Kouri GP, Bravo J, et al. Dengue hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic study. Am J Trop Med Hyg 1990; 42(2):179–184. 37. Halstead SB. Etiologies of the experimental dengues of Siler and Simmons. Am J Trop Med Hyg 1974; 23(5):974–982. 38. Innis BL. Antibody responses to dengue virus infection. In: Gubler DJ, Kuno G, eds. Dengue and Dengue Hemorrhagic Fever. Wallingford, UK and New York: CAB International, 1997:221–243. 39. Calvert AE, Huang CY, Kinney RM, et al. Non-structural proteins of dengue 2 virus offer limited protection to interferondeficient mice after dengue 2 virus challenge. J Gen Virol 2006; 87(pt 2):339–346. 40. Kurane I, Meager A, Ennis FA. Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity. J Exp Med 1989; 170(3):763–775. 41. Bukowski JF, Kurane I, Lai CJ, et al. Dengue virus-specific crossreactive CD8þ human cytotoxic T lymphocytes. J Virol 1989; 63(12):5086–5091. 42. Samuel MA, Diamond MS. Pathogenesis of West Nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion. J Virol 2006; 80(19):9349–9360. 43. Stephens HA, Klaythong R, Sirikong M, et al. HLA-A and -B allele associations with secondary dengue virus infections correlate with disease severity and the infecting viral serotype in ethnic Thais. Tissue Antigens 2002; 60(4):309–318. 44. Sakuntabhai A, Turbpaiboon C, Casademont I, et al. A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet 2005; 37(5):507–513.

Chapter 56: Vaccine Strategies to Prevent Dengue 45. Fernandez-Mestre MT, Gendzekhadze K, Rivas-Vetencourt P, et al. TNF-alpha-308A allele, a possible severity risk factor of hemorrhagic manifestation in dengue fever patients. Tissue Antigens 2004; 64(4):469–472. 46. Guzman MG, Kouri G, Valdes L, et al. Epidemiologic studies on dengue in Santiago de Cuba, 1997. Am J Epidemiol 2000; 152(9): 793–799. 47. Burke DS, Nisalak A, Johnson DE, et al. A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 1988; 38(1): 172–180. 48. Vaughn DW, Green S, Kalayanarooj S, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000; 181(1):2–9. 49. Guzman MG, Kouri G, Martinez E, et al. Clinical and serologic study of Cuban children with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Bull Pan Am Health Organ 1987; 21(3):270–279. 50. Halstead SB, Nimmannitya S, Cohen SN. Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J Biol Med 1970; 42(5):311–328. 51. Murgue B, Roche C, Chungue E, et al. Prospective study of the duration and magnitude of viraemia in children hospitalised during the 1996–1997 dengue-2 outbreak in French Polynesia. J Med Virol 2000; 60(4):432–438. 52. Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science 1988; 239(4839):476–481. 53. Halstead SB. Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 2003; 60:421–467. 54. Huang KJ, Yang YC, Lin YS, et al. The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol 2006; 176(5): 2825–2832. 55. Avirutnan P, Punyadee N, Noisakran S, et al. Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J Infect Dis 2006; 193(8): 1078–1088. 56. Halstead SB, Lan NT, Myint TT, et al. Dengue hemorrhagic fever in infants: research opportunities ignored. Emerg Infect Dis 2002; 8(12):1474–1479. 57. Kliks SC, Nimmanitya S, Nisalak A, et al. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg 1988; 38(2): 411–419. 58. Pengsaa K, Luxemburger C, Sabchareon A, et al. Dengue virus infections in the first 2 years of life and the kinetics of transplacentally transferred dengue neutralizing antibodies in Thai children. J Infect Dis 2006; 194(11):1570–1576. 59. Goncalvez AP, Engle RE, St Claire M, et al. Monoclonal antibodymediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proc Natl Acad Sci U S A 2007; 104(22):9422–9427. 60. Kliks SC, Nisalak A, Brandt WE, et al. Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg 1989; 40(4):444–451. 61. Stephenson JR. Understanding dengue pathogenesis: implications for vaccine design. Bull World Health Organ 2005; 83(4):308–314. 62. Blanc G, Caminopetros J. Contribution a l’etude de la vaccination contre la dengue. Bull Acad Natl Med 1929; 102(24):1–4. 63. Simmons JS, St John JH, Reynolds FHK. Experimental studies of dengue. The Philippine J Sci 1931; 44:1–252. 64. Innis BL, Eckels KH. Progress in development of a live-attenuated, tetravalent dengue virus vaccine by the United States Army Medical Research and Materiel Command. Am J Trop Med Hyg 2003; 69(6 suppl):1–4. 65. Monath TP, Kanesa-Thasan N, Guirakhoo F, et al. Recombination and flavivirus vaccines: a commentary. Vaccine 2005; 23(23): 2956–2958.

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57 Vaccination Against the Hepatitis C Virus Sergio Abrignani and Raffaele De Francesco INGM, Istituto Nazionale Genetica Molecolare, Milano, Italy

Michael Houghton Epiphany Biosciences, Inc., San Francisco, California, U.S.A.

INTRODUCTION Hepatitis C virus (HCV) is a remarkably successful blood borne virus that establishes a chronic infection in about 70% of the infected individuals. An estimated 3% of the world’s population, about 170 million people, is chronically infected by HCV (1). Acute infection is generally asymptomatic, making early diagnosis very infrequent. Twenty percent of chronically infected persons will develop liver cirrhosis, and liver failure or hepatocellular carcinoma occurs in up to 3% to 4%. Consequently, HCV infection is the most common indication for liver transplantation, accounting for 40% to 50% of liver transplants. However, liver transplantation is not a cure for HCV, as virus recurrence is universal. Although the transmission of HCV has declined substantially (2,3), an efficacious HCV vaccine would be of great public health benefit. In fact, the pool of asymptomatic chronic HCV carriers who represent an infectious reservoir will remain substantial for many years and approximately 35,000 new infections still occur annually in the United States alone. Moreover, certain modes of transmission will continue for the foreseeable future, such as injection drug use, which now represents the most frequent mode of transmission of HCV. Because of the high rate of chronic infection following acute HCV infection and the relative efficacy of the therapies currently available, prevention of new infections would be a cost-effective strategy for control of the disease. An efficacious HCV vaccine would be of potential benefit to all persons at risk for coming in contact with contaminated blood. This includes health care workers, hemodialysis patients, those with diseases requiring frequent blood products, IV drug users (IVDUs), and inmates of correctional facilities. Considering IVDU, an HCV vaccine might be broadly given to adolescents considered at risk for later drug use. Although the risk of sexual transmission of HCV is low, it may be reasonable also to recommend vaccination to the sexual partners of infected individuals. Eventually, a safe and efficacious HCV vaccine could be recommended for widespread general use in adolescents. An HCV vaccine should either prevent infection altogether or at least prevent the development of chronic infection following acute infection. Although preventing initial infection by providing ‘‘sterilizing immunity’’ would be ideal, experiments in chimpanzees indicate this will be difficult to achieve, but it may also be unnecessary. The very great majority of acute

infections are asymptomatic and without clinical consequences, and it is the manifestations of chronic HCV infection that lead to the clinically evident disease. Thus, a vaccine that allowed only a ‘‘transient infection’’ (either subclinical or of limited acuity), while preventing the development of chronic HCV infection could be as beneficial as one that provided sterilizing immunity. Theoretically, a vaccine that did not accomplish either could still be beneficial if it prevented or delayed the development of progressive liver disease, such as cirrhosis. However, practically, it would be difficult to demonstrate within a reasonable time frame the benefits of an HCV vaccine intended to modify the natural history of disease rather than to prevent infection. Another essential requirement for an HCV vaccine is that it must protect against the major circulating genotypes (Fig. 1). An HCV vaccine that demonstrated narrow genotype-specific efficacy might require geography-specific limitations in its use and would be difficult to use in practice. However, an efficacious HCV vaccine might exhibit a gradation of efficacy such that it might prevent infection by heterologous subtypes at a lower but still clinically significant level compared to more closely related subtypes. Proving efficacy of the vaccine in humans also represents a significant challenge, since accessing groups at high-risk of HCV infection is no longer a simple task. With the near elimination of post-transfusion hepatitis C by donor screening, other high-risk groups suitable for efficacy testing can have inherent difficulties such as lack of compliance (IVDUs), low incidence of infection (health care workers), lack of supporting infrastructure (in many developing countries where incidence of infection is high), and ethical issues (in prisoner populations where prevalence and incidence of infection are both high). However, some of these cohorts have been used successfully in the past (e.g., in the case of testing hepatitis B vaccines) and so these obstacles should not be insuperable.

THE VIRUS HCV is an enveloped positive-stranded RNA virus classified as member of the Hepacivirus genus within the Flaviviridae family (4). The 10 kb HCV RNA genome contains a single open reading frame encoding a polyprotein of about 3000 amino acids, flanked by highly structured 50 and 30 untranslated regions (UTRs), required for RNA replication and translations (5). Translation is mediated through an internal ribosome entry site (IRES)

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Figure 1 Approximate HCV prevalence and genotype distribution. Source: From Ref. 3.

situated within the 50 UTR of the RNA genome (6). A hepatocyte specific cellular micro-RNA, mir-122, stimulates HCV RNA translation by enhancing the association of ribosomes with the viral RNA at an early initiation stage (7). Thus, miR-122 is believed to contribute to HCV liver tropism at the level of translation. Following translation of the positive-stranded RNA genome, a polyprotein precursor is generated that is cleaved proteolytically into the mature structural and nonstructural proteins (8) (Fig. 2). The viral nucleocapsid protein core (C) and the envelope glycoproteins (E1 and E2), found in the aminoterminal third of the polyprotein, represent the structural components of the viral particle. The structural proteins are separated by the nonstructural proteins by p7, a short integral membrane protein believed to be an ion channel of the viroporin family. p7 is required for efficient assembly and release of infectious virions. The polyprotein junctions C/E1, E1/E2, and E2/p7 are processed co-translationally by signal peptidase cleavage. In addition, the action of signal-peptide peptidase is required to release the E2 signal peptide from the core protein. The remainder of the polyprotein encodes enzymes and accessory factors that take part in the replication of the HCV RNA genome and possibly participate in late events in the viral life cycle, such as viral particle packaging and egress. Thus, NS2 is a cysteine protease that acts in concert with the N-terminal third of NS3 to catalyze the cleavage at the NS2/NS3 site junction. In addition, NS2 is also involved in an early stage of virion morphogenesis. NS3 is a multifunctional protein with a serine protease domain located in the N-terminal third and an RNA helicase domain in the C-terminal two-thirds. NS3 forms a heterodimeric complex with the serine protease cofactor NS4A. The NS3-4A serine protease complex is responsible for the

proteolytic maturation of the NS3-5B region. Moreover, the NS3-4A protease complex has been implicated in the proteolytic inactivation of Trif and Cardif (9,10), two adaptor proteins crucial for sensing the viral invasion by the cell innate immune system. NS4B is an integral membrane protein that contributes to the formation of the membrane-associated replication complex. NS5A is a phosphoprotein that plays a crucial role in RNA replication and in virus assembly. Finally, NS5B is the viral RNA-dependent RNA polymerase. The NS proteins associate together on the cytoplasmic side of the endoplasmic reticulum (ER) membrane, where they presumably form the viral RNA replication complex (11). In addition to the viral proteins described earlier, a 2/þ1 ribosomal frame-shift has been observed to occur around residue 11 of the C protein, resulting in the synthesis of the frameshift (F) or alternative reading frame protein (ARFP) (12). Although the basic charged character of the F/ARFP protein may also imply a nucleocapsid-like function, its actual function is currently unknown. HCV infection is a highly dynamic, with a half-life in the order of a few hours and an estimated 1012 virions being turned over every day in a chronically infected individual (13). This impressively high replication activity, together with the lack of proofreading activity by the error-prone viral RNA-dependent RNA polymerase, is the basis for the observed high genetic variability of HCV. The 50 and 30 noncoding regions of the RNA genome are highly conserved as is the C gene encoding the nucleocapsid protein; in contrast, the rest of the viral genes/ proteins exhibit considerable heterogeneity. On the basis of the genetic distance among different isolates, six major genotypes and many subtypes have been distinguished phylogenetically. Genotypes may vary by 30% to 35% in their nucleotide sequence, whereas subtypes may differ by 15% to 20% (14).

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Figure 2 HCV genes and gene products. (A) The structure of the HCV genome and polyprotein. The polyprotein processing scheme is shown as follows: closed circles refer to signal peptidase cleavage sites; the open circle refers to the signal peptide peptidase cleavage site; the open arrow refers to the NS2-3 protease cleavage site; filled arrows refer to NS3-4A protease cleavage sites. (B) The topology of HCV proteins with respect to the endoplasmic reticulum membrane. Source: From Ref. 8.

Moreover, HCV circulates within an infected individual as a number of closely related but distinct species, termed ‘‘quasispecies’’ (15).

Models to Study HCV The major known site of HCV replication is the hepatocyte, but it is not clear how many hepatocytes in an infected liver support productive HCV infection. HCV has also been reported to infect other cell types, most notably B cells and dendritic cells (16–20). However, the matter of extrahepatic HCV infection remains controversial. The lack of a readily available laboratory animal susceptible to HCV infection has hampered studies targeting the interaction between the host immune cells and the sites of infection. The only animal specie that is susceptible to HCV infection is the chimpanzee. The animals currently available to the research community, however, are chronically infected chimpanzees, since very few naı¨ve individuals have been made accessible due to the declining availability of these animals, as well as ethical concerns. In alternative to the chimpanzee model, immune-deficient mice can be genetically manipulated to obtain destruction of the liver transplanted with human hepatocytes and, in turn, experimentally infected

with HCV (21). The usefulness of this model, however, is limited by the high mortality of the mouse strains and by the lack of a functional immune system. Many attempts were made to propagate HCV in cell culture, but these attempts proved unsuccessful for many years. Alternative surrogate model systems were therefore established to elucidate various aspects of the HCV life cycle. The description of autonomously replicating subgenomic HCV replicons was the first demonstration of in vitro HCV replication (22). Replicons are autonomously replicating RNA molecules carrying all the genetic elements necessary for self-replication (the NS3-NS5B region flanked by the 50 and 30 UTRs). Such replicons are able to self-replicate in the human hepatoma cell line Huh-7, and replication is dependent on adaptive mutations (23,24). This system provided the first opportunity for studies of efficient HCV RNA replication in vitro, but it is of limited relevance for studies related to the development of a vaccine, especially with regard to the possibility of developing an in vitro virus-neutralizing antibody assay. Important advances in this respect were provided by the development of systems that recapitulated in vitro the viral entry process. Currently, there are two methods available to study HCV entry. The first one is based on a retrovirus in which HCV E1 and E2 substitute for the endogenous retroviral

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envelope proteins (25,26). The resulting pseudo-particle (HCVpp) is capable of infecting cells with a specificity that is dictated by the HCV envelope proteins. Since the HCVpp can express a range of HCV glycoproteins originating from different isolates and genotypes, it represents the ideal platform for the establishment of virus-neutralizing antibody assay (27). The second method to study viral entry was provided by the recent development of a cell culture system for HCV propagation, resulting in the production of significant levels of infectious virus particles. This development built on the unique growth capacity of strain JFH1 (genotype 2a) (28–32). The viral particles produced by this system (termed ‘‘HCVcc’’ for cell-culturederived HCV) were shown to be infectious in a chimpanzee, confirming the authenticity of the HCV particles generated during cell culture propagation. By development of JFH1based intergenotypic recombinants containing Core, envelope protein 1 and 2 (E1, E2), p7, and nonstructural protein 2 (NS2) from different viral isolates, a panel of culture systems for all major HCV genotypes was established (33). Unfortunately, at present this system is limited to selected viral isolates, often containing adaptive mutations that replicate efficiently in a sub-clone of the hepatocarcinoma cell line HuH-7, and do not produce high titers of virus. Nevertheless, the availability of a bona fide in vitro infection model has catalyzed a much greater understanding of viral entry and morphogenesis processes.

Virions, Cellular Receptors, and Morphogenesis Despite the advent of an authentic in vitro infectivity system, viral particles have not been conclusively visualized, and the virion structure has not yet been elucidated. On the basis of the limited available data, the HCV particle is thought to have a diameter of about 50 nm. By analogy to related viruses, HCV virions are thought to consist of an icosahedral lattice of E1–E2 heterodimers bound to a cell-derived lipid bilayer membrane surrounding a nucleocapsid built from multiple copies of the core protein which in turn package the genomic RNA. The HCV core protein is a dimeric a-helical protein containing two domains, D1 and D2, with distinct properties. The N-terminal D1 domain is hydrophilic and highly basic and has been implicated in RNA binding and oligomerization. The hydrophobic D2 domain directs the transfer of fully matured core from the ER membrane to lipid droplets (LDs) (34). LDs are intracellular storage organelles found in all eukaryotic organisms. They consist of a core of neutral lipid, comprising mainly triacylglycerols and/or cholesterol esters, surrounded by a monolayer of phospholipids and associated proteins. It has been speculated that the interaction of core with LDs might affect lipid metabolism of the infected hepatocyte, contributing to the development of liver steatosis that is often associated with chronic HCV infection (35). Moreover, a critical role is emerging for LDs as a cellular organelle crucial implicated in virion morphogenesis (see the following text). E1 and E2 are type I membrane proteins composed of a C-terminal transmembrane domain and a large N-terminal ectodomain (36). During translation, the ectodomains of E1 and E2 are directed to the ER lumen and the transmembrane domains are inserted in the ER membrane, to which E1 and E2 remain anchored. During their biogenesis, E1 and E2 form a functional noncovalent dimer that is believed to be the protein building block of the viral envelope (37–39). The N-terminal 30 amino acids of E2 contain the hypervariable region termed ‘‘HVR-1.’’ The E2 HVR-1 has been shown to contain antibody-binding

viral neutralizing epitopes using the chimpanzee infection model (40–45). The high variability of this region is thus thought to contribute to HCV escape from the immune response. In addition, E1 and E2 contain, respectively, several highly conserved N-glycosylation sites that have been implicated in protein folding, modulation of viral entry, and protection against neutralization (46,47). HCV is invariably found in association with low-density and very low-density lipoproteins (LDL and VLDL), resulting in the so-called lipo-viro-particles (48). It has been suggested that interaction of host lipoprotein with cell surface receptors provides a route of binding and entry for lipoprotein associated virus, which may be independent of viral glycoproteins (see the following text) and which may render the virus resistant to antiviral antibody mediated neutralization. Thus, cellular surface molecules such as the LDL receptor (LDL-R) and glycosaminoglycans (GAGs) have been implicated in facilitating virus binding and uptake (49–51), possibly by enriching the virus at the cell surface via nonspecific binding to the HCV-associated lipoproteins prior to specific receptor interaction. None of these molecules, however, has conclusively been demonstrated to be strictly required for HCV entry. Conversely, three cellular surface proteins have unequivocally been shown to be essential (co)-receptor for HCV: the tetraspanin family member CD81, scavenger receptor class B type I (SR-BI), and claudin-1 (52). CD81 and SR-BI were identified on the basis of their ability to bind directly to E2 (53,54). Antibodies or small-interfering RNA against CD81 or SRBI reduce infectivity of HCVpp or HCVcc in cell culture (25,29,30,32,55–57). Moreover, ectopic expression of CD81 is sufficient to confer infection susceptibility to a CD81-deficient hepatoma cell line (25,30,32). Coexpression of CD81 and SR-BI in cells of nonhepatic origin, however, is not sufficient for infection. Claudin-1, a tight junction component highly expressed in the liver was recently identified as an additional essential HCV entry cofactor (58). CLDN1 is believed to act late in the entry process, after virus binding and interaction with the HCV coreceptor CD81. In addition to being required for HCV entry in hepatoma cell lines, CLDN1 is also able to confer susceptibility to HCV when ectopically expressed in some nonhepatic cells. Cell types were found, however, that remained nonpermissive to HCV entry despite expression of CD81, SR-BI, and claudin-1, indicating that at least an additional host cell factor may be required as HCV coreceptor. The observation that both CD81 and SR-BI bind E2 suggests that E2 is directly implicated in viral receptor recognition and entry. The E2 hypervariable N-terminal region (HVR1, see the following text) has been identified as a main determinant for binding to SR-BI, whereas several noncontiguous sites downstream of HVR1 have been implicated in binding CD81, suggesting a conformational binding site for the latter (co-) receptor. The role of E1 in viral entry is less well established, and there is more limited evidence of neutralizing determinants within E1. Interestingly, another tight junction protein, namely occluding (OCLN), has been recently reported to be another essential part of the HCV receptor complex (59,60). This discovery highlights the importance of the tight junction complex in the viral entry process. Altogether, expression of OCLN, CD81, SR-BI, CLDN1 was shown to render murine cells susceptible to infection by HCVpp. Although the murine versions of SR-BI and CLDN1 can substitute for their human counterparts, both OCLN and CD81 need be of human origin to allow efficient viral entry. The identification of OCLN as the ‘‘missing’’ factor required for HCV entry is likely to represent a

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crucial advance toward the development of a small animal model for HCV infection. Following virus-receptor interaction on the cell surface, HCV particles are taken up clathrin-dependent endocytosis and membrane fusion occurs in the early endosomes (61,62). It has also been recently observed that, at least in vitro, HCV infection can be transferred to naı¨ve cells via direct cell-cell contact in a way that is independent of CD81 but dependent on claudin-1 (63). Details of how the virus enters the cell in cell-cell transmission mode are currently unknown, as is the significance of cellcell HCV transmission in vivo. However, it is important to bear in mind that such CD81 independent virus transmission may offer a strategy for HCV to escape the effect of neutralizing antibodies. Specific alterations of the ER membrane, termed ‘‘membraneous web’’ were identified at the site of RNA replication by ultrastructural studies of cells containing HCV replicons (64). The membranous web is composed of vesicles of 80 to 180 nm in diameter that resemble the ‘‘sponge-like inclusions’’ previously described in the liver of HCV-infected chimpanzees. The vesicles contain the viral RNA replication machinery. The expression of high amounts of viral replicative proteins (65) on the ER membranes induces a state of ER stress in the cell. ER stress normally results from the accumulation of unfolded proteins in the ER, in response to which cells activate an intracellular pathway known as the unfolded protein response. ER stress may explain why cells expressing HCV proteins have a lower major histocompatibility complex (MHC) class I cell surface expression. HCV-infected cells may thus have a deficiency in their ability to present viral antigens (66). Due to the recent availability of a bona fide HCV in vitro infection system, the processes of virion morphogenesis and exit from the cells are beginning to be elucidated. One striking discovery has been that cytoplasmic LDs are organelles critically implicated in HCV virion production (67). Thus, mutations that are known to impair localization of the core protein on the LDs have been also found to negatively impact virion production by HCV-infected cell lines (68). In addition, it has been observed that the core protein localized at the surface of LDs is able to recruit HCV replication complexes that are in turn associated to the ER membrane, primarily through the interaction with the NS5A protein (67,69,70). This recruitment induces the juxtaposition of core-coated LDs and ER membrane bound replication complexes generating a docking platform where it is believed that newly synthesized viral RNA is transferred from the replication complex to the LD-bound core to facilitate the first steps of nucleocapsid packaging and possibly the interaction of the nucleocapsid with the ER-bound E1E2 heterodimer. HCV particle production is inhibited by agents that block VLDL assembly (71). In light of these observations, it has been hypothesized that HCV replication complexes recruit LDs to secure the lipid stock necessary for the production of lipo-viro-particles (48).

NATURAL IMMUNITY AND IMMUNE CORRELATES Studies in the chimpanzee challenge model and of multiply exposed humans demonstrate that there is significant natural immunity against HCV. In one chimpanzee study (72), an animal was infected by intrahepatic administration of an infectious RNA derived from HCV strain HCV-1 (of the 1a genotype). This genotype is the most common in the United States (Fig. 1). Following resolution of the ensuing acute infection and disappearance of viremia, the animal was shown to be resistant to an

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intravenous rechallenge with homologous virus. No viremia was observed following rechallenge, indicating that sterilizing immunity was generated by the original infection. When rechallenged subsequently with a heterologous 1a strain, the animal experienced very transient, minimal viremia unlike the substantial viremia observed in control animals. Resolution of the infection in the challenged animal was confirmed by showing the subsequent absence of viral RNA from the blood and liver. Furthermore, when rechallenged again with heterologous 1b genotype, the most common worldwide (Fig. 1), only a transient viremia was observed prior to disappearance of the virus from plasma and the liver (72). Very similar results were obtained in separate chimpanzee studies in which animals were challenged and then rechallenged intravenously (IV) with different infectious viral inocula. Only transient viremia occurred when animals that recovered initially from a 1a infection were rechallenged with either a heterologous 1a strain or a heterologous 1b strain (73,74). Opposite results have been obtained in another chimpanzee study (75) in which protection against chronic HCV infection was observed only after rechallenge with homologous, but not heterologous, genotypes. Moreover, it has been recently reported that previously infected chimpanzees are not consistently protected against reinfection or persistent infection after reexposure to the identical HCV strain (76). In man, evidence of cross-protective immunity has also been reported in a prospective study of IVDUs from the United States. Strikingly, the incidence of persistent viremia in IVDUs who had recovered from a previous infection was 12-fold lower than that in IVDUs who had not experienced a previous infection, as shown using multivariate analyses (77). As seen in the chimpanzee studies, peak viral loads were substantially higher (by almost 2 logs) in the first-time infections as compared with the reinfections. Also, HIV coinfection produced persistent HCV infection in all cases, indicating the role of the immune response in HCV recovery (77). Intriguingly, it has now been shown that HIV patients coinfected with HCV have lowered peripheral immune responses to HCV as compared with patients with HCV monoinfections, even in the absence of severe CD4þ T-cell depletion (78). Interestingly, it has been recently reported that the reduced risk of HCV persistence in IVDUs previously recovered from HCV infection correlated with T-cell responses, and prolonged antigenic stimulation appears to be required to maintain humoral responses (79). Collectively, these chimpanzee and human data provide evidence for the existence of immunity to HCV and, importantly suggest the existence of cross-protective immunity within and between commonly occurring HCV clades. It is important to note, however, that not all reinfections in IVDUs are resolved without progressing to chronicity (77), indicating that natural immunity to HCV is not complete and not as effective as for the hepatitis A and B viruses. It should also be mentioned that earlier studies in the chimpanzee model have concluded a lack of protective immunity to HCV (80). This apparent contradiction may be due to the earlier studies having measured immunity more in terms of sterilizing immunity rather than the ability to prevent the development of chronic infection. It was first noted that early and broad MHC class II– restricted CD4þ T helper responses to HCV are associated with recovery from acute, symptomatic infections of man (Fig. 3) (81,82). Subsequently, using the valuable chimpanzee model again, it was shown that recovery could occur because of an early and broad MHC class I–restricted CD8þ cytotoxic lymphocyte (CTL) response to the virus (83). This association of

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Figure 3 Proliferative CD4þ T-cell response of the first sample in the acute phase of disease to recombinant HCV proteins (HCV– NS3, –NS4, –NS5, and –core) of peripheral blood mononuclear cells (PBMCs) from 38 patients with acute hepatitis C. Patients are grouped according to the final outcome of disease in self-limited hepatitis C (SL, n ¼ 20) and patients with chronic evolution (C, n ¼ 18). Results are shown as SI–3H-thymidine incorporation of antigenstimulated PBMCs (cpm)/unstimulated control (cpm). Values higher than three are considered significant. All patients with self-limited disease displayed a significant proliferative T-cell response against at least one of the viral proteins, while patients with chronic evolution mounted no or only transient antiviral T-cell responses. NS3 and NS4 revealed the most frequent and most vigorous responses. In four patients, the proliferative response against NS5 was not tested in the first sample. Source: From Ref. 81.

HCV-specific CD4þ T helper and CD8þ CTL responses with resolution of acute infection has been suggested in many human studies (11,84–90). Another study sheds further light on the potential mechanism of recovery from clinically asymptomatic, acute infections of man. Shortly following infection, activated HCV-specific CD8þ T cells appeared in the peripheral blood that were associated with an increase in serum alanine aminotransferase (ALT) levels (signifying liver damage), and with a small decrease in viral RNA levels. These activated CD8þ cells did not secrete g-interferon. Shortly afterward, a 5log reduction in viral load occurred commensurate with the appearance of HCV-specific, g-interferon-secreting CD8þ cells. This large reduction in viral load was not accompanied by an increase in serum ALT levels, suggesting that a noncytolytic, viral clearance mechanism mediated by g- interferon might be operative (Fig. 4) (88). This conclusion is consistent with the demonstrated anti-HCV activity of g-interferon in cell cultures containing HCV replicons (90). In contrast, individuals who develop chronic, persistent infection show weaker cellular immune responses to the viruses that are not maintained over time (85–89,91–94) (Fig. 4). The importance of HCV-specific, cellular immune responses is further reinforced by data from several studies showing that memory T-cell responses to the virus can be detected in long-term convalescent individuals (93), in

Figure 4 HCV-specific T-cell responses in subject I during asymptomatic, resolving acute HCV infection. (A) Course of infection. (B) Percentage of CD8þ lymphocytes that were tetramer-positive at each time point. CD8þ T-cell responses were tested directly ex vivo using HLA-A2 tetramers complexed with five different HLA-A2 restricted epitopes. CD8þ T-cell responses against two epitopes [NS3 1073 and NS3 1406 (VA)] were detectable. (C) Percentage of NS3 1406 (VA)-specific CD8þ T cells expressing the activation marker CD38. (D) Percentage of CD8þ T cells that produce IFN-g in response to HCV NS3 1406 (VA) (black bars) and NS3 1406 (SG) (white bars). (E) Proliferative CD4þ T-cell responses against core, NS3, NS4, and NS5 are shown as the sum of all positive stimulation indices. (*) Sum of all specific stimulation indices is 56. Source: From Ref. 88.

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nonviremic and HCV antibody-negative, healthy family members of HCV patients (95), and in other individuals who lack HCV antibody and RNA but who may have been exposed to the virus earlier (96). However, further work is necessary to define the relative roles of HCV-specific CD4þ and CD8þ T cell responses in protection as well as the mechanism of action. The kinetics of induction of HCV-specific T-cell responses to multiple epitopes may be crucial in determining the outcome of infection, because the ability of the virus to mutate and thus evade CD8þ T-cell responses has been demonstrated convincingly in the chimpanzee model (97). The latter study suggests that if a multi-specific T cell response is made early in infection, then it is harder for the virus to mutate several epitopes simultaneously and, therefore, more likely to result in resolution of infection. Host and viral factors involved in determining the breadth, strength, kinetics, and decline (91,92) of HCVspecific cellular immune responses will deserve much attention in the future. In this regard, certain MHC class I and class II alleles are associated with recovery in humans (98–100). The role of antienvelope antibody in resolution of HCV infection is unclear at present. Initially, it was found that nearly all chronically infected humans have significant antibodies to gpE1 and gpE2, as measured in ELISA assays, and that there was not a clear relationship between the induction of these antibodies and resolution of acute infection (101). These findings were extended in other studies showing that in man and chimpanzees, it was more likely to find antienvelope antibody (as measured in ELISA assays) in chronically infected individuals than in resolvers of acute infection (102–104). This contrasts with the situation found for many infectious agents, for example, the hepatitis B virus, where antienvelope antibody is a strong correlate of immunity. Evidence for the generation of neutralizing antibody comes from several studies showing the efficacy of human Ig preparations, derived from numerous donors, in preventing the transmission of HCV following blood transfusion (105), liver transplantation (106), and between sexual partners (107). Chimpanzees that received human Ig prior to experimental viral challenge also showed a clear inhibition of acute hepatitis and viremia throughout the lifetime of the antibodies (108). When combined with clinical evidence for worse HCV-associated disease in hypogammaglobulinemics (109,110), the data clearly suggest that HCV infection does induce neutralizing antibody. Why then is there a lack of correlation between antienvelope antibody and the outcome of infection? Several possibilities exist; first, it has been shown that antisera raised against the hypervariable N-terminal region of gpE2, (HVR1) can neutralize the infectivity of homologous virus in the chimpanzee model (111). Preliminary studies have reported that the early induction of anti-E2 HVR1 may correlate with resolution of acute infection in humans (112,113). Many studies have also suggested that this region mutates readily in response to the specific, humoral immune response, thus evading the binding of anti-E2HVR1 (40,42,45). If this is a principal neutralizing domain of the virus, then it is possible to imagine how the virus might evade the antibody response. This might also explain the apparent, protective efficacies of complex human Ig preparations, if they contain highly plural anti-E2 HVR1 antibodies. A second possibility is that HCV infects cells not only by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81 and occludin, but also through direct cell-to-cell transmission (63). Unfortunately, this last mode of transmission is shielded from neutralizing

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antibodies (114). These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission. Now that HCV has been propagated in cell culture, assays for viral-neutralizing antibody are available. Moreover, HCVpp have been used to show that patients not only have antibody that can neutralize the infectivity of such pseudoparticles but that such antibody cross-neutralizes pseudoparticles derived from many different HCV genotypes (115–117). This suggests that broad cross-neutralizing antibody to HCV may exist and could be exploited in vaccine strategies. Furthermore, the recent application of the HCVpp infectivity assays to investigate immune correlates of protection are beginning to indicate that such ‘‘neutralizing’’ antibody when present, may also be associated with recovery from acute infection, at least in some cases (118). In more recent studies, availability of cell culture viruses (JFH1 intergenotypic recombinants) with envelope proteins of the six major HCV genotypes permitted crossneutralization studies in vitro. 1a and 4a serum cross-neutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses, demonstrating a clustering of genotypes that, if confirmed, will be of importance for future development of passive and active HCV immunoprophylaxis strategies (119). Interestingly, using a human liver-chimeric mouse model, it has been found that human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates protect against heterologous HCV quasispecies challenge in vivo. This result provides evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection in vivo and suggests that a prophylactic vaccine against HCV may be achievable (120).

VACCINE STRATEGIES AIMS Vaccine approaches to date have included the use of adjuvanted recombinant polypeptide subunits in attempts to prime viral-neutralizing antibody to the envelope glycoproteins 1 and 2 (gpE1 and gpE2), as well as MHC class II–restricted CD4þ T helper and MHC class I–restricted CD8þ CTL responses to various viral proteins. Both types of T cells can secrete antiviral cytokines like g-interferon, and CD8þ CTLs have the potential to kill infected cells. It is difficult to prime CD8þ CTLs using polypeptide subunit vaccines, although certain adjuvants are capable of eliciting such responses (121,122). Various forms of plasmid DNA vaccines are also being explored to elicit HCV-specific humoral and cellular immune responses to encoded antigens, which, by virtue of being newly synthesized in the cytosol of transfected cells, can be particularly effective at priming CD8þ CTLs. DNA vaccines can also include immunostimulatory CpG-containing motifs capable of activating antigen-presenting dendritic cells leading to stimulation of innate immune responses [like the synthesis of type 1 interferons and natural killer (NK) cells ] as well as adaptive B- and T-cell responses to vaccine antigens. Various live attenuated or defective viral vectors expressing HCV genes are also being investigated, since improved vaccine immunogenicity can result from more efficient expression and delivery of HCV antigens including the targeting of antigen-presenting cells in some cases. The use of various

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prime/boost immunization modes and regimens are also being explored to optimize vaccine immunogenicity and potency.

Adjuvanted, Recombinant HCV Protein Vaccines A vaccine comprising the recombinant gpE1/gpE2 heterodimer combined with an oil/water microemulsified adjuvant has been tested for immunogenicity in rodents (123) and for efficacy in the chimpanzee model (124–127). The heterodimer was derived from HeLa cells infected with a recombinant vaccinia virus expressing a C–gpE1–gpE2–p7–NS20 gene cassette derived from the HCV-1 strain (1a genotype). Following cleavage by host signal peptidase in the ER, gpE1 and gpE2 translocate into the lumen of the ER where they remain associated together and tightly anchored via type 1 transmembrane domains located at the C termini of both molecules (128). Following extraction in nonionic detergent and affinity purification using GNA-lectin (that specifically binds the high mannose chains found on the heterodimer), the purified subunit proteins were used to immunize naı¨ve chimpanzees along with the adjuvant. Generally, 30 to 40 mg of the subunits were administered intramuscularly (IM) on approximately months 0, 1, and 6, and a homologous viral challenge was given intravenously, two to three weeks after the third immunization. Encouragingly, of seven animals receiving the vaccine, five were completely protected against the challenge, with no signs of viral infection in any of the assays, including sensitive RT-PCR assays for viral RNA (Fig. 5) (124). These five apparently sterilized animals were the highest responders to the vaccine in terms of elicited titers of anti-gpE1/gpE2. Sterilization did not correlate with antibody titers to E2 HVR1 but did correlate with the titer of antibodies that block the binding of recombinant E2 to the virus receptor CD81 (53,129). In addition, the two lowest responders that became infected following challenge eventually resolved the acute infection without becoming chronic carriers (Fig. 5) (53,124,126,127,129). In contrast, most control animals become chronically infected following viral challenge. Given that the prime, practical goal of an HCV vaccine would be to prevent the development of chronic infection following exposure to the virus, this study provided much encouragement for the development of an effective vaccine challenge. This work has been extended to address the key question of whether the vaccine protects against experimental challenge with a heterologous 1a viral strain (HCV-H strain, another member of the 1a genotype common in the United States) (125). It has been shown that out of a cumulative total of 10 vaccinees challenged with the heterologous HCV-H strain, while all 10 animals experienced acute infections, only 1 developed chronic, persistent infection. In contrast, most control animals challenged with HCV-H become chronic carriers (Table 1). Interestingly, antibodies alone were not sufficient for protection, indicating that vaccine-mediated T-cell responses and/or the host’s immune response to HCV replication following challenge are also involved in the protection observed. Another group has also immunized a chimpanzee with insect cell-derived recombinant gpE1 and gpE2 (from HCV strain N2), in addition to a peptide spanning the E2 HVR1 (from HCV strain #6). gpE1 and gpE2 were each expressed and purified separately and lacked the C-terminal transmembrane anchors. The E2 HVR1 peptide was conjugated to keyhole limpet hemocyanin, and Freund’s adjuvant was employed to augment immune responses. Their conclusion, from a series of immunizations and challenges with HCV strain #6 in the same

Figure 5 HCV-1 challenge of vaccinated and control chimpanzees. Control and immunized chimpanzees (L, laboratory for experimental medicine and surgery in primates; W.S., white sands) were challenged with 10 CID50 of HCV-1. The presence (solid boxes) or absence (open boxes) of hepatocyte ultrastructural changes observed in the EM is indicated. The approximate relative levels of HCV-1 RNA detected in plasma using RT-PCR assays are reflected by sizes of the shaded circles (open circles denote borderline positives; minus signs denote undetectable levels). The results of RT-PCR assays of liver and peripheral blood leucocyte (PBL) extracts are recorded as either þ or . The arrows in the alanine aminotransferase axes indicate the mean þ3.75 SD of prechallenge values; open vertical arrows denote time of subunit vaccine administration, and the solid vertical arrow denotes viral challenge on week 0. Abbreviations: IU/L, international units per liter; RT, reverse transcriptase; EM, electron microscope; ALT, alanine aminotransferase. From Ref. 124.

animal, was that sterilization was dependent on high anti-E2 HVR1 titers rather than anti-gpE2 or anti-gpE1 titers (130,131). The outcome of challenging with other viral strains was not reported, however. It should also be noted that insect-derived gpE2 (truncated at the C terminus) has been shown to bind poorly to the HCV receptor, CD81, as compared with gpE2 derived from mammalian cells (81). The latter group also showed that the full-length gpE1/gpE2 heterodimer, derived from mammalian cells, bound to CD81 with high affinity, as

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Table 1 Summary of Outcome of Experimental Challenges of Chimpanzees Immunized with Recombinant gpE1/gpE2 Group Homologous HCV-1-challenges Heterologous HCV-H challenges Totals

Total

Acute infections

Chronic infections

12 10 9 14 21 24

7 10 9 14 16 24

2 (17%) 7 (70%) 1 (11%) 8 (57%) 3 (14%) 15 (63%)

Adjuvanted gpE1/gpE2 Unimmunized controls Adjuvanted gpE1/gpE2 Unimmunized controls Adjuvanted gpE1/gpE2 Unimmunized controls

p ¼ 0.03 p ¼ 0.04 p ¼ 0.002

Typically, animals were immunized with 30 to 80 mg gpE1/gpE2 in oil/water adjuvants on months 0, 1, and 7 approximately, followed by intravenous challenge on month 8 with 10 to 100 infectious doses of HCV-1 or HCV-H. Circulating levels of viraemia were measured using RT-PCR assays for HCV genomic RNA for at least one-year post challenge. p values refer to chronic carrier rates between controls and vaccines. Source: From Ref. 125.

did intracellular forms of gpE2, truncated at the C terminus. However, secreted forms of gpE2 were not effective at binding CD81. Because deglycosylation of secreted gpE2 restored CD81-binding activity, it appears that the addition of complex carbohydrate during secretion either masks the CD81-binding site or changes the conformation to an inactive form (132). The physiological significance of these findings is unclear at present, because little is known on the nature of the virion carbohydrate. Intracellular, C-terminally truncated forms of gpE2 have been shown to be immunogenic in human volunteers, although efficacy in the chimpanzee challenge model has not yet been demonstrated (M. Houghton and S. Abrignani, unpublished data). However, the gpE1/gpE2 heterodimer is generally considered to be a more native reflection of the HCV virion than either ectodomain alone and, as mentioned earlier, has demonstrable prophylactic efficacy in the chimpanzee. Therefore, recombinant gpE1/gpE2 represents an encouraging human vaccine candidate. Some work has been directed to the synthesis of viruslike particles (VLPs) by expressing the structural genes in different cells. In insect cells, expression of the C–gpE1–gpE2 gene cassette has been reported to result in the generation of 40 to 60-nm VLPs within cytoplasmic cysternae (133). Following partial purification, these VLPs appear to be immunogenic in small animals and, as such, represent a potential vaccine candidate for humans (134). Similar-sized VLPs have been observed in the process of budding into the lumen of the ER, following expression of the same gene cassette in mammalian cells using a Semliki Forest viral vector (135). HCV gene expression was observed to induce convoluted membranes or membranes similar to those seen in infected livers. However, some viral or host factor is limiting the release of the VLPs into the lumen and from being secreted into the cell media. Because the HVR1 region of gpE2 has been shown to possess viral neutralizing epitopes and to be highly mutable, attempts have been made to select for a cross-reacting version using phage display of mimotopes. Rabbit antisera raised against one mimotype was highly cross-reactive with the E2 HVR1 of many viral isolates and reacted to discontinuous epitopes (136). Such mimotypes or consensus E2 HVR1 peptides may be valuable components of an HCV vaccine. In this regard, a consensus E2 HVR1 peptide sequence has been fused to the B subunit of cholera toxin and expressed in plants through the use of a tobacco mosaic viral vector. Crude extracts were then administered intranasally to mice, which then generated cross-reactive E2HVR1 antibodies that were capable of capturing virions (137). Recombinant C protein is also being studied as a potential component of an HCV vaccine for several reasons. First,

broad HCV-specific CD4þ and CD8þ T-cell responses, including those of the C protein, are associated with recovery from HCV infection (83,85,86,88,89). Second, the C protein is the most conserved HCV polypeptide and contains CD4þ and CD8þ epitopes that are highly conserved among the different HCV genotypes, which should therefore facilitate the generation of cross-protective immunity (138,139). Third, recombinant C protein has been shown to self-assemble into particles (140) with concomitant high immunogenicity in animal models. Substantial CD4þ T cell priming and high anti-C antibody titers have been obtained in mice and sheep (141,142). In general, immunization with adjuvanted polypeptide subunits does not result in cross-priming, a process in which the antigen is deposited within the cytosol of antigen-presenting cells and processed peptides then placed on the MHC class I presentation pathway, resulting in stimulation of CD8þ CTLs. However, through the use of the immune stimulating complex (ISCOM) adjuvant, a particulate adjuvant comprising cholesterol, phospholipid, and naturally occurring saponins, it has been possible to prime strong CD8þ CTL activity (and CD4þ activity) in rhesus macaques, using a recombinant C antigen derived from E. coli (Fig. 6) (122). CTLs to at least two epitopes were identified. On the basis of these data, a phase I study was performed (143). In this study, a phase I placebo-controlled, dose escalation clinical trial was designed to evaluate immunogenicity of the HCV core Iscomatrix vaccine in healthy individuals. The 30 subjects received three immunizations of HCV core Iscomatrix vaccines or placebo vaccine on days 0, 28, and 56. The vaccines contained 5, 20, or 50 mg HCV core protein with 120 mg Iscomatrix adjuvant. Antibody responses were detected in all but one of the participants receiving the vaccine. However, CD8(þ) T-cell responses were only detected in two of the eight participants receiving the highest dose. The results of this study suggest that the development of a protein-based HCV vaccine capable of priming cross-reactive HCV-specific CD8þ T-cell responses in man will require further investigation. There are numerous CD4þ and CD8þ epitopes that are conserved among the different HCV genotypes. These reside within the various HCV-encoded virion and nonstructural proteins (138,139,144). The inclusion of a multiplicity of these epitopes will facilitate the vaccine-mediated generation of broad cellular immune responses to the virus, which could then result in the elicitation of cross-protective immunity against different HCV genotypes. One such approach involves the assembly of ‘‘HCV polytope vaccines’’ consisting of a consecutive sequence of these conserved HCV T cell epitopes in the form of a recombinant polypeptide or DNA vaccine. Focusing the immune response on a collection of highly conserved epitopes that can be presented by diverse human MHC

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Figure 6 Longevity of the CTL responses primed by vaccination with recombinant HCV core in ISCOMs adjuvant. Peripheral blood mononuclear cells from rhesus macaques DV037 (A) and BB232 (B) were restimulated in vitro with the epitopic peptide 121 to 135. After CD8þ enrichment, cells were tested for cytotoxic activity against autologous B-LCLs sensitized with the epitopic peptide 121 to 135 (6) or an irrelevant peptide (0). (C), Freshly isolated PBMCs from DV037 51 weeks after its last immunization (two left panels) or in vitro restimulated PBMCs from the same time point (two right panels) were restimulated for 12 hours with peptide 121 to 135 or a control peptide and stained for surface CD8 and intracellular IFN-g and TNF-a. Lymphocytes were gated by side versus forward scatter light, and then for CD8-PerCP. Plots show log fluorescence intensity for TNF-a-FITC and IFN-g-PE. Abbreviations: CTL, cytotoxic lymphocyte; HCV, hepatitis C virus; ISCOM, immune stimulating complex; LCLs, lateral collateral ligament; PBMCs, peripheral blood mononuclear cells; IFN-g, interferon gamma; TNF, tumor necrosis factor; FITC, Fluorescein isothiocyanate; PE, Phycoeritrine. Source: From Ref. 122).

class I and class II antigens may optimize the generation of strong, cross-protective immunity and possibly avoid the ‘‘dilution’’ of the immune response to variable, more mutable HCV epitopes.

HCV DNA Vaccines The discovery that naked DNA or RNA administered intramuscularly or intradermally (ID) results in expression of encoded antigens, and the elicitation of specific humoral and

cellular immune responses has opened up a new area of vaccinology (145–148). Advantages of using a DNA vaccine relate to the ease and cost of manufacture, the gene-mediated synthesis in vivo of native and often complex protein structures (that would otherwise be difficult to produce in the form of recombinant proteins), good stability (that renders the use of DNA vaccines particularly suitable to the needs of developing countries), and the ready ability to employ multiple genes, gene cassettes, or plasmids to elicit broad immune responses against heterogeneous pathogens such as HIV, HCV, and malaria.

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Another important feature relates to the ability of DNA vaccines to stimulate CD8þ MHC class I–restricted CTL responses via the endogenous synthesis of proteins de novo in the cytosol. Disadvantages, however, can include a weaker potency as compared with other vaccine formulations as well as safety issues revolving around their potential to integrate into the host genome, thereby increasing the risks of mutagenesis and carcinogenesis. Additional safety issues may also be of concern, depending on the infectious agent under study. In the case of HCV, relatively long-term expression of the C gene has been reported to exert multiple pathogenic, oncogenic, and regulatory functions in transfected cells and animals. Its role in inducing steatosis and hepatocellular carcinoma in transgenic mice has been documented (149,150), as has its ability to copromote cellular transformation in vitro (151). There have been numerous other reports demonstrating the ability of the C gene product to bind to a member of the tumor necrosis factor (TNF)-a superfamily (152), modulate apoptosis (151), induce oxidative stress (153,154), activate cellular and viral promoters (155), and affect other regulatory functions (156). While the pathogenic significance of these findings in infected humans, if any, remains to be determined, it may be prudent in the meantime to omit the C gene from a potential HCV DNA vaccine (although the intermittent use of a recombinant C polypeptide subunit vaccine is unlikely to pose such a safety risk). Similarly, the 50 terminal region of the NS3 gene (encoding the protease domain) has been linked with transformation of cells and carcinogenesis in nude mice (157). Most of the work performed so far with HCV DNA vaccines has been performed in small animals, with very little done using nonhuman primates. This is important because, while most DNA vaccines are immunogenic in mice and other small animals, such results are harder to reproduce in higher animals. Furthermore, even the rhesus macaque is a questionable model for immunogenicity of DNA vaccines in humans. Many studies have immunized mice with plasmids expressing the envelope genes of HCV, generally using the immediateearly CMV promoter and intron to achieve high expression levels. Accordingly, the generation of humoral and cellular immune responses to gpE2 has been widely reported, including cross-reactive anti-gpE2 antibodies between subtypes 1a and 1b (158–164). The use of a DNA vaccine encoding intracellular forms of gpE2, rather than secreted forms, has been emphasized to elicit anti-gpE2 antibodies capable of preventing the interaction between gpE2 and the HCV receptor CD81 (132). Boosting DNA-primed mice with recombinant gpE2 has been reported to elicit higher anti-gpE2 titers than repeated DNA or protein immunizations (165). Immunogenicity of gpE2 DNA vaccines has also been demonstrated in rhesus macaques (158). As with DNA vaccines targeting other infectious agents, the mode of injection drastically alters the immune response. Administration of a gpE2 DNA vaccine by gene gun, in which DNA is physically administered intraepithelially on gold microparticles, resulted in anti-gpE2 titers that were 100 times higher than if delivered by needle intramuscularly (162). This increase in vaccine potency should translate to the use of lower doses in humans, thus lessening safety concerns. Other methods to improve the potency of DNA vaccines include the application of an electric field at the site of DNA injection (socalled electroporation). In the case of a gpE2 DNA vaccine, electroporation led to 10-fold increases in expression levels and in concomitant anti-gpE2 responses. The latter also included cross-reactive anti-E2HVR1 antibodies that were not obtained

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without the use of electroporation. This technique also resulted in significant increases in gpE2-specific CD4þ T helper and CD8þ CTL responses (166). However, the potential of electroporation to increase the rate of integration of the DNA vaccine into the host genome remains to be evaluated. Other improvements include the formulation of DNA vaccines into particles, thereby increasing the uptake by antigen-presenting dendritic cells with corresponding increases in immunogenicity (167). Lipid formulations of DNA that result in improved transfection efficiency in vivo as well as better uptake by dendritic cells have been shown to be part of an encouraging immunization regimen for rhesus macaques against simian-human immunodeficiency virus (SHIV) challenge and have applications to HCV and other infectious diseases (168). Only one small HCV DNA vaccine study in the chimpanzee model has been reported so far. In an attempt to optimize immunogenicity, the ectodomain of gpE2 (aa 384– 715) was fused to the CD4 C-terminal, transmembrane region (TMR) that facilitated sequestration of the encoded gpE2 glycoprotein to the outer cell surface, rather than being anchored in the lumen of the er via the use of the homologous TMR. Ten milligrams of DNA were administered using a bioinjector into the quadriceps of two animals on weeks 0, 9, and 23, followed by experimental challenge with homologous, monoclonal virus three weeks later. Humoral and cellular immune responses to the vaccine were observed in only one animal, but following challenge, viremia was lowered as compared with a control animal and hepatitis occurred earlier, as a result of the primed immunity. Importantly, both vaccinees resolved their acute infections quickly, whereas the control, unvaccinated animal became chronically infected following viral challenge. This result is promising, but further studies are warranted due to the small number of animals and because one of the vaccinees had already experienced an experimental HCV infection, prior to vaccination, that would have conferred immunity (169). In order to recapitulate the broad T-cell responses associated with protective immunity against HCV infection, many groups are investigating DNA vaccines capable of priming HCV-specific CD4þ and CD8þ T cell responses to many HCV gene products. A DNA vaccine encoding NS3, NS4, and NS5 not only primed broad and specific antibodies, CD4þ T helper and CD8þ CTL responses, but also conferred protection to the immunized BALB/c mice against challenge with syngeneic, SP2/0 myeloma cells expressing NS5 (170). Coexpression of the granulocyte macrophage-colony stimulating factor (GM-CSF) cytokine gene has also been shown to augment cellular immune responses to these NS gene products when administered as a bicistronic plasmid to buffalo rats (171). Many studies have been conducted with DNA vaccines containing the HCV C gene, as this encodes the most conserved viral protein and is known to contain important T -cell epitopes. Immunogenicity in mice has been reported (172–175) and, in addition, the coadministration of either IL2 or GM-CSF has been shown to augment humoral and cellular immune responses to C in mice (172). On the contrary, coimmunization with a plasmid expressing IL4 resulted in the elicitation of a Th0 phenotype and a concomitant suppression of C-specific CTLs (172). The use of transgenic mice expressing human HLA-A2.1 has also shown the ability of NS3 DNA vaccines to induce specific CTLs to the same immunodominant epitope observed in humans (176). Finally, it is also noteworthy that defective RNA vaccines have been shown to be very effective at protecting against flaviviral infections (177). Conferring good protective immunity

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in the absence of integration into the host genome, this approach has great promise for HCV and other infectious diseases.

HCV Vaccines Using Disabled Viral Vectors The use of a defective or attenuated viral vector to deliver vaccines has several potential advantages. Firstly, a wide tropism of the host vector leads to the efficient delivery of the vaccine genes and encoded antigens. Preferably, this tropism includes antigen-presenting cells leading to a very effective priming of the immune response, thereby requiring only one immunization for long-lasting immunity. The use of a vector already used as a vaccine itself, offers further obvious advantages with respect to manufacturing, distribution, and user acceptance. Finally, many vectors allow the insertion of multiple genes thus facilitating the induction of a broad, crossprotective immune response, particularly useful against heterogeneous agents such as HCV. One such promising approach for HCV has been the use of an attenuated rabies viral vector into which either the HCV gpE1–gpE2–p7 gene cassette was inserted, or just the ectodomain of gpE2 linked to the CD4, C-terminal TMR, and cytoplasmic domain. In the case of the latter construction, recombinant rabies virions were produced that actually contained the hybrid gpE2 within the virion. Virions expressing gpE1–gpE2–p7 were immunogenic in mice eliciting CTL responses to gpE2 (178). Similarly, defective Semliki Forest virions containing the HCV NS3 gene produced long-lasting NS3-specific CTLs after one immunization in mice transgenic for human HLA-A2.1 (179). As observed in HCV-infected patients, the immune response was directed to one immunodominant epitope within NS3. Defective, recombinant adenoviruses expressing the HCV C–gpE1–gpE2 gene cassette have also been shown to prime HCV-specific CTLs in mice immunized intramuscularly, although the induction of anti-gpE1/ gpE2 antibodies required further immunization with purified gpE1/gpE2 glycoproteins (180). Replication-defective adenoviruses expressing C and gpE1 also primed long-lasting, specific CTL responses in mice (181). Recombinant canary pox viruses, expressing an HCV gene cassette containing C–gpE1–gpE2–p7– NS2–NS3, elicited HCV-specific humoral and cellular immune responses in mice, although the optimum immunization regimen required first priming with a plasmid DNA expressing the HCV genes prior to boosting with the recombinant canary pox virus (182). It has been showed that vaccination with adenoviral vectors and electroporated plasmid DNA encoding the HCV non structural region NS3 to NS5B, protected chimpanzees from acute hepatitis induced by challenge with a heterologous virus differing from the vaccine sequence by more than 13% at the amino acid level (183). Four out of five vaccinated chimpanzees developed a cross-reactive T-cell response against the challenge virus that resulted in a low viremic state, although no difference was observed compared to the control group with respect to the number of animals that did not proceed into the chronic carrier state. It is likely that in the absence of neutralizing antibodies, cellular responses are unable to eradicate infection, thereby making this vaccine approach interesting to investigate as a therapeutic add-on to other therapies. A recent interesting approach has been the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding

HCV proteins (184). In this study, mice were immunized with defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345), and strong CD8(þ) T-cell responses but low CD4(þ) T helper responses to these HCV gene products were detected. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4(þ) T helper responses but no CD8(þ) T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4(þ) T helper responses but no CD8(þ) T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4(þ) and CD8(þ) T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans to recapitulate all of the cellular and humoral immune response in an ideal vaccine regimen.

SUMMARY Data indicating the existence of natural immunity against the HCV and vaccine efficacy in the chimpanzee challenge model allow optimism for the development of at least a partially effective vaccine against this heterogeneous pathogen that is responsible for much of the chronic liver disease around the world. A few years ago, prospects for effective vaccination against HCV were considered remote because of the high propensity of this virus to promote chronic persistent infections, evidence that convalescent humans and chimpanzees could be readily reinfected following reexposure as well as the considerable genetic heterogeneity of this virus. The situation today can be more optimistic for several reasons. First, we now know that the spontaneous eradication of virus occurring in a consistent fraction of acute infections is associated with specific immune responses to the virus. Recapitulation of such immune responses by appropriate vaccination therefore becomes a realistic option. Second, clear evidence for at least some natural immunity has emerged in both humans and chimpanzees. These studies have shown that convalescent humans and chimpanzees are protected from chronic infection against reexposure to virus in the majority of cases, even against very divergent viral strains. Some issues surrounding clinical development of a prophylactic HCV vaccine remain to be solved. Not only is it difficult to identify the appropriate at risk population to enroll in an efficacy trial for a preventive HCV vaccine, but it may also be very difficult to conclude an efficacy trial designed to measure prevention of chronic infection. As discussed earlier, the great majority of acute infections are asymptomatic and without clinical consequences, and it is the manifestations of chronic HCV infection that lead to the clinically evident disease. Thus, a vaccine that allowed only a ‘‘transient infection’’ (either subclinical or of limited acuity), while preventing the development of chronic HCV infection could be as beneficial as one that provided sterilizing immunity. Indeed, vaccine efficacy data from the chimpanzee challenge model indicate that is possible to prevent the progression to chronic infection in vaccinees. However, this endpoint, in the absence of correlates of

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immunity, makes it very complicated to organize efficacy clinical trials. In fact, studies on acute HCV patients have shown that acute infections treated with interferon within the first three months of onset are eradicated in almost all patients (185). As the chronic state of the infection is defined starting from six months after onset, it becomes very difficult to conclude an efficacy trial. When an infected vaccinee is detected, interferon treatment will have to be started without delay and it will be impossible to establish whether or not the infection will become chronic. Therefore, it is likely that the approval of a hepatitis C vaccine by regulatory agencies will require innovative processes, such as releases, on the basis of the animal efficacy rule and phase IV follow ups. The U.S. Food and Drug Administration (FDA) Animal Efficacy Rule (186) provides a mechanism for licensure when human efficacy challenge-studies are not feasible or ethical, such as for smallpox or anthrax. Considering that HCV is the first cause of primary liver cancer, if a vaccine is successfully developed, an important cause of global morbidity and mortality will be controlled and even in countries with a relatively low incidence of infection, the vaccine will be reasonably cost-effective when used in the general population.

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58 Vaccines Against Respiratory Syncytial Virus and Parainfluenza Viruses Alexander Schmidt, Emmalene Bartlett, Anne Schaap-Nutt, Peter L. Collins, and Brian R. Murphy Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland, U.S.A.

Ruth Karron Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, U.S.A.

GENERAL CONSIDERATIONS The Viruses and the Diseases They Cause Respiratory syncytial virus (RSV) and three human parainfluenza viruses (PIV1, PIV2, and PIV3) are responsible for up to 50% of lower respiratory tract illness (LRTI) leading to hospitalization of infants and young children, including pneumonia, bronchiolitis, bronchitis, and croup (1,2) (Table 1). RSV is the most frequent cause of a child’s first LRTI, and approximately 1% of any birth cohort is hospitalized with RSV bronchiolitis or pneumonia, often at an age as young as one to six months. PIV3 is less frequent but can cause the same disease spectrum as RSV and just as early in life, whereas PIV1 and PIV2 disease is seen more commonly in children over six months of age. Laryngotracheobronchitis or croup is the signature disease of PIV1, and PIV1 is the single most common cause of croup. A likely immunization sequence employing RSV and PIV vaccines would be administration of RSV and PIV3 vaccines together as a combined vaccine that would be given two or more times, with the first dose administered at or before one month of age, followed by a bivalent PIV1 and PIV2 vaccine at four and six months of age. RSV and PIV3 are also significant causes of respiratory illness in immunocompromised patients and in the elderly, and a separate set of vaccines will likely be needed for these target groups because of their seropositive status. RSV and PIV infection usually starts in the upper respiratory tract (URT) from where it may or may not progress to the LRT. In the immunocompetent host, virus replication is generally limited to the respiratory tract. RSV and the PIVs infect both ciliated cells in the LRT and alveolar cells, but not the basal cells within the bronchial epithelium (3,4). RSV pathology is dominated by obstruction of small airways by inflammatory cell debris, edema, and external compression from hyperplastic lymphoid follicles without extensive epithelial cell damage (5). RSV and the PIVs are members of the Paramyxoviridae family (subfamily Pneumovirinae and Paramyxovirinae, respectively) in the Order Mononegavirales, that is, they are nonsegmented negative sense single stranded RNA viruses. A typical RSV virion (Fig. 1A) consists of a nucleocapsid that is surrounded by a lipid envelope. Three proteins are embedded

in this envelope: the G protein, which is the main mediator of viral attachment, the fusion protein (F), which mediates fusion of the virus envelope with the cell membrane, and the small hydrophobic protein (SH), which has no defined function. The matrix protein (M) is thought to be located on the inner face of the envelope and is important in virion morphogenesis. The nucleocapsid itself consists of the RNA genome and the proteins of the polymerase complex: the large polymerase protein (L), the nucleoprotein (N), the phosphoprotein (P), and M2-1 protein, which supports processivity of the polymerase during transcription. The genomes of RSV (Fig. 1B) and the PIVs (PIV3 is shown in Fig. 1C) are approximately 15.5 kb in length and are organized into gene units that are separated by short intergenic sequences (IG, bold in Fig. 1C). Gene start (GS) sequences and gene end (GE) sequences direct initiation and termination of mRNA transcription for each gene unit. Transcription occurs in a sequential manner in which the viral polymerase scans the genome from the 30 end and transcribes each gene unit in turn. Polymerase drop-off between gene units leads to a transcriptional gradient in which gene units closer to the 30 end of the genome are transcribed more efficiently than downstream genes. The virion structure and genome organization of the PIVs are similar to that of RSV but PIV does not encode the SH, NS1, NS2, and M2 proteins. In addition, the viral attachment proteins of the PIVs have a neuraminidase function and are referred to as hemagglutinin-neuraminidase (HN) proteins (Fig. 1C). Both RSV and the PIVs encode for a number of non-essential proteins that play an important role in inhibiting the host’s innate immune response, for example, the NS1 and NS2 proteins of RSV, the C protein family of PIV1 and PIV3, and the V protein of PIV2 and PIV3.

Immunity to RSV and PIV RSV and PIV are sensitive to the interferon (IFN) arm of the innate immune response and have developed measures to inhibit this first line of the host’s defense (6–8). The T-cell and humoral arms of the adaptive immune response, including CD8þ and CD4þ T cells as well as IgG and IgA antibodies,

Chapter 58: Vaccines Against RSV and PIV Table 1 Contribution of RSV, PIVs, and Influenza to Pediatric Hospital Admissions for Acute Respiratory Disease Study (reference) Design Study duration N enrolled, age Virus identification RSV PIV1 PIV2 PIV3 Influenza A

Washington, D.C., 1958–1976 (1)

Germany, 1999–2001 (2)

Prospective, single-center 1958–1976 3523–5104, <6 yr Virus culture þ serology 23% 6% 3% 12% 3–7%

Prospective, multicenter 1999–2001 592, <3 yr PCR 38% 3% 1% 5% 4%

Abbreviations: RSV, respiratory syncytial virus; PIV, parainfluenza virus.

contribute to viral clearance and to protection against disease. Virus neutralizing antibodies present in serum or mucosal secretions are known correlates of protection against disease in humans, and CD8þ and CD4þ T cells have been shown to provide short term protection against RSV in mice (9–14). Neutralizing antibodies are directed against the attachment glycoprotein and the fusion glycoprotein of these viruses. Transudation of neutralizing serum IgG antibodies to the luminal surface of the LRT confers significant protection against RSV and PIV disease and is the basis for the successful

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use of an anti-RSV F monoclonal antibody to prevent serious RSV disease in early infancy (15). Mucosal neutralizing IgA antibodies, which are associated with protection against infection as well as disease upon reinfection, are best induced by two or more infections with wild-type (wt) virus. Therefore, two or more mucosal vaccinations will likely be needed to efficiently induce this important arm of the immune response (16). Recognizing that it is difficult to prevent infection with RSV or PIV, the major goal of immunizations is to induce a level of immunity that will restrict viral replication sufficiently well to prevent severe disease associated with the first or second wt virus infection.

Obstacles in Vaccine Development A number of obstacles have to be overcome to develop effective RSV and PIV vaccines (Table 2). First, immunity induced by infection with wt virus often is insufficient to block symptomatic reinfection. Even following repeated infections with wt virus, prevention of infection is achieved only in a minority of pediatric subjects. Most people experience multiple URT infections (URTI) with RSV and PIV, and reinfection with homologous RSV can occur within a single season (17). However, illness upon subsequent infection is generally less severe than after first infection, that is, disease is restricted to the URT or is only mild in the LRT with subclinical involvement of bronchioles or alveoli. Therefore, immunization against RSV and PIV, like vaccination against poliovirus or rotavirus, will likely

Figure 1 Electron micrograph of an RSV particle (A) with indicated structural and nonstructural viral proteins and their functions. The genome organization of RSV (B) and PIV3 (C) are shown, not drawn to scale. Gene units are depicted as rectangles, and letters identify the encoded protein(s). For PIV3, the gene end, intergenic (IG, bold) and gene start sequences are shown for the P-M gene junction. These cisacting sequences are highly conserved and govern initiation and termination of mRNA transcription. Abbreviations: RSV, respiratory syncytial virus; PIV, parainfluenza virus; le, leader; tr, trailer.

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Table 2 Obstacles to RSV and PIV Vaccine Development 1. Immunity and immunopathology . Complete protection against infection and disease is difficult to achieve . Live attenuated vaccines are less immunogenic than wild-type virus . Multiple doses of vaccine are needed to provide durable mucosal immunity . Formalin-inactivated RSV vaccine induced enhanced disease 2. Young age of vaccinees . Decreased immune response in first 6 months of life . Maternal antibodies to RSV and PIV can decrease immune response to vaccine . Difficult to achieve a balance between attenuation and immunogenicity for live attenuated virus vaccines . Clinical trials for vaccines for use in infancy proceed slowly . Age-related temporal association with sudden infant death syndrome (SIDS) and reactive airway disease (RAD) . Interference with routine vaccines needs to be excluded 3. Virus specific factors . Highly infectious viruses able to infect partially immune subjects . Limited or no cross-protection (RSV A vs. RSV B)—five vaccines needed (RSV A, RSV B, PIV1, PIV2, PIV3) . Lack of highly permissive animal models . Poor growth and limited physical stability (RSV only) Abbreviations: RSV, respiratory syncytial virus; PIV, parainfluenza virus.

require multiple doses of vaccine to achieve durable immunity. Second, most of the severe RSV and PIV3 disease occurs in young infants aged one to six months (18), the target population for a RSV and PIV3 vaccine, but infants infected with wt virus in the first six months of life generate less antibody to the viral surface glycoproteins than older infants (19,20). In addition, younger infants have a less diverse B cell repertoire, their antibody affinity maturation is less efficient, and T-cell help is limited (19,21–24). Thus, the immature immune response of the young infant is a significant obstacle for a vaccine designed to protect that age group. Third, maternal antibodies present in the young infant can modify the response to immunization. These transplacentally acquired RSV and PIV neutralizing serum antibodies reduce the immunogenicity of parenterally administered subunit vaccine, vectored vaccine, and wt virus infection through a poorly defined immunological mechanism termed antibody-mediated immune suppression (25–27). Fourth, for RSV, two antigenic subgroups (designated subgroup A and B) cocirculate and are only 25% related antigenically (18). A second infection within the same season is often caused by virus belonging to the heterologous subgroup, indicating that antigenic diversity is responsible in part for the high frequency of second infections with RSV, and more importantly, for LRT disease upon second infection (18). Therefore, a total of five separate vaccines will be needed to protect against RSV and PIV disease, two for RSV (RSV subgroup A and RSV B) and three for PIV (PIV1, PIV2, and PIV3). Fifth, during RSV vaccine trials in the 1960s, immunization of infants and young children with an inactivated RSV vaccine unexpectedly potentiated RSV disease following subsequent natural wt RSV infection (28,29). This observation, in the context of comparable observations for inactivated measles virus vaccine, mandates that vaccine development for RSV proceeds with great caution in the pediatric population. In addition, infants will be vaccinated at an age when sudden infant death syndrome (SIDS) occurs, and this will raise

a concern for the use of any topical RSV or PIV vaccine even though these viruses are not thought to be causal agents. This is not unlike the concern regarding the occurrence of intussusception in recipients of a live rotavirus vaccine. This and other reasons given in Table 2 make the development of a live virus vaccine for use in early infancy especially challenging. In summary, successful vaccines against RSV and PIV must (i) be immunogenic in young infants, even in the presence of maternally-acquired serum antibodies; (ii) protect against LRTI following first infection with wt virus; (iii) induce resistance to both subgroup A and B strains of RSV; and (iv) not induce a immune response that can lead to enhanced RSV or PIV disease during subsequent natural infection. Despite these obstacles, it should be possible to successfully immunize the target pediatric population against RSV and PIV.

VACCINES FOR RSV AND PIV Nonreplicating Virus Vaccines for RSV Nonreplicating virus vaccines include inactivated whole virus vaccines, virus-like particles, subunit vaccines such as purified glycoproteins, and DNA or RNA vaccines that express one or more protective antigens of RSV or PIV. Inactivated whole RSV vaccines for seronegative infants are not currently being evaluated in clinical trials, but instructive information was derived from their previous use. These vaccines have been extensively reviewed elsewhere and will not be discussed in detail here (30). Formalin-inactivated RSV (FI-RSV) was evaluated in the 1960s and resulted in disease potentiation (increased frequency and severity of bronchiolitis and pneumonia) in vaccinees following infection with wt RSV (28,29) (Table 3). At least two factors are thought to have contributed to disease potentiation in FI-RSV vaccinees. First, FI-RSV failed to induce a significant level of resistance to RSV replication because (i) the antibodies induced by FI-RSV had greatly diminished neutralizing activity and (ii) FI-RSV failed to induce a protective CD8þ T-cell response. Second, FI-RSV induced a nonprotective but disease enhancing Th2 type CD4 þ T-cell response (40) and was also associated with immune complex deposition in the airways (41). Thus, RSV replicated in FI-RSV vaccinees without significant immunological restriction, but the T cell–mediated inflammatory cell response was accelerated and augmented, and this translated into an increase in the frequency and severity of bronchiolitis and pneumonia. Enhanced disease has not been observed with natural RSV or PIV infection, reinfection, or with live attenuated RSV or PIV vaccines, and was not seen in seropositive subjects immunized with FI-RSV or RSV subunit vaccine (18). Thus, disease potentiation is not associated with replicating RSV vaccines, probably because they induce highly functional antibodies, CD8þ T cells, and a more Th1-biased response. A number of subunit RSV vaccines have been evaluated in preclinical trials, and several of them have progressed into clinical trials (Table 3). Most of these candidate vaccines consist of either one or both of the viral surface glycoproteins that mediate membrane fusion (F) or virus attachment (G), or parts thereof. Some of these candidate vaccines contained adjuvants such as aluminum hydroxide or aluminum phosphate, CpG nucleotides, monophosphoryl lipid A (MPL), saponins, or oil-in-water emulsions, while others were conjugated to bacterial toxins or formulated as immunostimulating complexes (31,34). The safety of many of these adjuvants for infants remains to be determined. Two observations will make

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Table 3 RSV Vaccines Evaluated in Clinical Trials Vaccine

Composition

Sponsor, clinical results (reference)

Program status

FI-RSV

Formalin-inactivated, concentrated whole RSV

Discontinued

PFP-1, PFP-2, PFP-3

F protein purified from RSV-infected Vero cells

FG

Recombinant fusion protein; (ectodomains of F and G) Coformulated purified F, G, and M proteins Part of the G protein (G2Na) fused to the albumin-binding domain of streptococcal G protein (BB) Biologically derived RSV mutants; live intranasal

NIAID; evaluated in infants and young children in the 1960s. Not protective; primed for enhanced disease in RSV-naı¨ve vaccinees (28,29) Wyeth; evaluated in adults, elderly adults, seropositive children and pregnant women. Safe and moderately immunogenic (31) GlaxoSmithKline; results of clinical studies not reported (32) Aventis-Pasteur; safe and immunogenic in adults (33) Pierre Fabre; evaluated in young adults and elderly adults; insufficiently immunogenic (34,35) NIAID and Wyeth; safe in adults but over- or under-attenuated in seronegative children and/or infants (20) University of Warwick; ts1C safe in adults and immunogenic in some individuals (36) NIAID and Wyeth (disconinued); NIAID and MedImmune RSV248/404/1030 DSH; safe and immunogenic in young infants; additional candidates being developed (19,37) Merck; evaluated in children with serum antibody to RSV; poorly immunogenic and not protective (38)

F, G, M BBG2Na Various cp, ts, and cpts mutants ts mutants 1B and 1C Recombinant cpts and deletion mutants

Biologically derived RSV mutants; live intranasal Recombinant RSV mutants prepared by reverse genetics; live intranasal

Wild-type RSV

Live

Intramuscular B/HPIV3-F

Recombinant B/HPIV3 expressing the RSV F protein

MedImmune; safe and immunogenic in adults (39)

Discontinued

Discontinued Active Discontinued

Discontinued

Discontinued Active

Discontinued

Active

Abbreviations: RSV, respiratory syncytial virus; PIV, parainfluenza virus; PFP, purified F protein; BBG2Na.

the use of subunit vaccines in seronegative infants challenging: first, like FI-RSV, subunit vaccines induce a high titer of RSVbinding (ELISA) antibodies that have low neutralizing activity; second, the enhanced disease seen with FI-RSV can be replicated in rodent models with subunit vaccines (42,43). Several purified F protein (PFP) vaccines (PFP-1, PFP-2, and PFP-3) have been evaluated by Wyeth Vaccines in clinical trials (31), but to our knowledge these clinical development efforts have been discontinued (Table 3). The fundamental weakness of PFP vaccines was their inability to induce a high titer of RSV neutralizing antibodies. PFP-3 (adjuvanted with aluminum phosphate), for instance, was found to be safe and immunogenic in seropositive children with cystic fibrosis, but the vaccine did not confer protection against RSV (44). Similarly, a significant increase in RSV neutralizing IgG titers was not observed following vaccination of pregnant women in their third trimester (45). These observations indicate that it is difficult to increase RSV antibody titers by immunization of young or adult seropositive individuals by a subunit vaccine. However, if subunit vaccines can be formulated to induce a high titer of neutralizing antibodies, they likely would have usefulness for immunization of the elderly, high-risk seropositive children, or immunocompromised persons that have previously been primed for an antibody response by natural infection. A nonreplicating protein vaccine probably would be acceptable for use in these groups because the disease potentiation that is associated with this type of vaccine has only been observed in RSV-naı¨ve individuals. Since it is apparently difficult to achieve a sustained increase in RSV antibody titers

in seropositive subjects, RSV vaccines to protect the elderly might have to be given annually, for example, together with the annual influenza virus vaccine, to provide coverage for a single RSV season. Sanofi-Aventis developed a subunit vaccine consisting of purified F, G, and M proteins that was found to be safe and immunogenic in healthy adult volunteers, but antibody titers were too short lived to provide long-term protection (33) (Table 3). The Institute Pierre Fabre generated a novel, bacterially expressed recombinant candidate vaccine by fusing the conserved central domain of the RSV G protein to the albuminbinding region of streptococcal protein G (Table 3). Although this candidate vaccine was found to be safe and immunogenic in phase 1 and 2 clinical trials, unexpected side effects such as purpura and type III hypersensitivity reactions occurred infrequently in phase 3 trials, and this halted further clinical development (34). DNA and RNA vaccines for RSV and PIV have not reached clinical trials yet and will not be considered here. In addition, non-living vaccines for PIV are not currently in clinical trials and will also not be further discussed.

Live Virus Vaccines Live virus vaccines either can be live attenuated RSV or PIV strains or can be live attenuated virus vectors that express RSV or PIV protective antigens. The live attenuated virus vaccines can also function as vectors to create multivalent RSV and PIV vaccines.

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Table 4 General Principles in Live Attenuated RSV and PIV Vaccine Development 1. Achieve balance between attenuation and immunogenicity in target population . RSVcpts248/404 is slightly under-attenuated in seronegative infants (20) . RSVcpts248/404/1030DSH is appropriately attenuated (19) . HPIV3cp45 is appropriately attenuated (46) . RSVcpts530/1009DNS2 is over-attenuated (37) 2. Maintain high infectivity . 100 HID50 are needed to infect >90% of vaccine recipients 3. Ensure replication to high titer in Vero cells . Virus needs to grow well in Vero cells so that vaccine can be manufactured economically and 100 HID50 can be delivered to the vaccinee 4. Use multiple doses since live attenuated virus vaccines induce less immunity than wild type 5. Multiple means to achieve attenuation are available . Cold passage . Chemical mutagenesis . Use of hr restricted viruses . Use of reverse genetics to generate att mutants or to increase immunogenicity . Identify attenuating point mutations . Combine point mutations to increase level of attenuation . Delete viral genes, e.g., interferon-antagonists such as NS1 gene of RSV . Move antigens promoter-proximal to increase immunogenicity 6. Genetic modifications help increase genetic and phenotypic stability of vaccines . Use of an alternate codon at site of an attenuating amino acid substitution . Delete one or two codons at site of an attenuating amino acid substitution . Delete gene or open reading frame . Substitution of an attenuating hr gene for a wild-type gene . Combine one or more attenuating mutations, especially ts and non-ts att mutations Abbreviations: RSV, respiratory syncytial virus; PIV, parainfluenza virus; hr, host-range; HID, human infectious dose; cp, cold passage; ts, temperature sensitive; att, attenuated.

General Principles Underlying the Development of Live Attenuated RSV and PIV Vaccines General principles common to the development of live attenuated RSV and PIV vaccines are summarized in Table 4. First and foremost, there is a delicate balance between attenuation and immunogenicity, and achieving this balance has proven very challenging. For RSV and PIV, the severity of acute respiratory illness correlates with the level of virus replication in the respiratory tract, and restriction of replication results in attenuation, that is, amelioration or absence of disease. However, reducing the magnitude of replication also reduces immunogenicity. Thus, the goal is to achieve the highest level of replication (and immunogenicity) of a live virus vaccine without the occurrence of any illness in the vaccinee. wt RSV reaches a peak virus titer of 105 plaque forming units (PFU)/mL of nasal wash fluid in ill subjects (47) whereas appropriately attenuated RSV mutants that do not cause disease in seronegative infants typically grow to a mean titer of approximately 102 to 103 PFU/mL (19). Similarly, wt PIV3 reaches a peak titer of >105 PFU/mL whereas an appropriately attenuated vaccine virus such as HPIV3cp45 reaches a mean peak titer of approximately 103 PFU/mL in seronegative infants (48). Fine-tuning the level of replication of

a vaccine virus to achieve an acceptable balance between attenuation and immunogenicity is therefore key to successful vaccine development. Examples of live attenuated viruses that represent a range of attenuation are indicated in Table 4. Second, it is important that a live attenuated vaccine retains high infectivity to induce protective immunity in almost all vaccine recipients. A useful measure of infectivity is the human infectious dose 50 (HID50), which is the dose, expressed as viral titer, that is required to infect 50% of vaccine recipients. As a general principle, one hundred HID50 should infect at least 90% of vaccinees. As one attenuates a respiratory virus, the HID50 might increase, and determination of the HID50 can therefore be useful during vaccine development. Also, live attenuated virus vaccines that are highly infectious and safe in infants will usually be poorly infectious (over-attenuated) in seropositive children and adults (37), and therefore they will not be useful immunogens in the seropositive population. It will be very difficult to develop a live attenuated virus vaccine that is highly infectious in seropositive children or adults and, at the same time, safe for seronegative infants who could become infected by contact with a seropositive vaccinee. For this reason, nonreplicating vaccines may provide the best alternative for immunization of older at-risk populations. Third, the attenuated vaccine virus needs to replicate efficiently in vitro, that is, it needs to grow to high titer in a cell line that is acceptable for vaccine manufacture, such as Vero cells. It is relatively easy using modern reverse genetic techniques to decrease the replication of RSV or PIV both in vitro and in vivo, but it is a challenge to attenuate a virus in vivo while maintaining a sufficiently high level of replication in vitro to support manufacture. As a minimum, live attenuated vaccine viruses need to replicate in Vero cells to titers significantly >100 HID50. Fourth, since live attenuated viruses are highly restricted in replication in the human respiratory tract (100- to 1000-fold lower titers than wt virus), they induce significantly less antibody than wt virus. The major means to compensate for this diminished immunogenicity is to give repeated doses of vaccine at defined intervals. One cannot increase immunogenicity with live RSV or PIV vaccines by increasing the quantity, or dose, of vaccine virus administered because immunogenicity is a function of the level of vaccine virus replication. The level of replication is determined by the attenuating mutations present in the vaccine virus and not by the dose administered. In other words, vaccine dose level affects the ‘‘take rate’’ of a vaccine, but not its level of replication in susceptible individuals nor its immunogenicity. Single doses of satisfactorily attenuated RSV and PIV vaccines have been found to be immunogenic in young infants (20,48), but a second dose of vaccine administered one to three months later can infect a large percentage of the vaccinees and further increase the magnitude of the immune response (48). Fifth, a variety of approaches can be used to attenuate RSV or PIV (Table 4). Serial passage of wt virus at suboptimal temperatures (cold passage, cp) is a traditional approach, and several RSV and PIV candidate vaccines have been generated in this way (46,49). Attenuated mutants generated by cp may exhibit a cold-adaptation (ca) phenotype, that is, they replicate more efficiently at low temperature (20–258C) than wt virus, or a temperature-sensitive (ts) phenotype, that is, they replicate inefficiently in vivo at physiological temperature. cp viruses can contain multiple attenuating (att) mutations, both ts and non-ts (50). A ts phenotype is desirable for a respiratory virus vaccine

Chapter 58: Vaccines Against RSV and PIV

because it allows the vaccine virus to replicate relatively efficiently in the nasopharynx (32–348C) but not in the LRT (378C). This restricted replication in the warmer LRT enhances the safety profile of a ts virus for the LRT. ts viruses can replicate efficiently at permissive temperature in cells used to manufacture the vaccine. ts viruses can also be generated by chemical mutagenesis, for example, by addition of a mutagen to the culture medium followed by selection of a mutant virus with a ts phenotype (Table 4). RSVcpts-248/404 is an example of a cold-passaged virus that was further attenuated by two rounds of chemical mutagenesis to generate a highly ts and highly attenuated virus (51). An entirely different approach to generate a vaccine virus is to use an antigenically related animal RSV or PIV that contains genes or sequences that restrict replication of the virus in humans. These attenuating genes or sequences confer a host-range (hr) att phenotype, that is, replication is efficient in the animal host of origin but restricted in humans. The use of bovine PIV3 (BPIV3) or bovine RSV (BRSV) to protect against human PIV3 or RSV are examples for the use of hr vaccines (52,53). Although BPIV3 replicates efficiently in Vero cells, it is highly attenuated in humans. It is believed that such hr phenotypes involve multiple genes or nucleotide sequences that contribute to attenuation (54). Therefore, the att phenotype of an hr vaccine virus should be phenotypically stable following replication in humans (55). Generation of infectious virus from genomic or antigenomic cDNAs, a technique known as reverse genetics, is a powerful new tool in the generation of attenuating mutations for use in live attenuated virus vaccines (Table 4). Reverse genetics can be used to define the contribution of individual point mutations or genes to the viral phenotypes (att, ca, ts, and hr). One can then design new recombinant viruses containing desired combinations of these attenuating elements, which can be evaluated to identify those with improved characteristics of attenuation and immunogenicity. Reverse genetics also allows for deletion of genes that are non-essential for replication in vitro such as those that mediate IFN antagonism. Deletion of IFN antagonists permits the host’s IFN response to respond to virus infection more vigorously and to restrict virus replication of the vaccine virus in the respiratory tract of the vaccinee. Fortunately, Vero cells lack functional type I IFN genes and thus these deletion mutants are able to replicate efficiently in this qualified cell line. Reverse genetics can also be used to increase the immunogenicity of vaccine viruses. Since promoter-proximal genes of paramyxoviruses (genes that are closer to the 30 end of the genome) are transcribed more efficiently than promoter-distal genes, expression of a protective antigen can potentially be increased by moving the respective gene closer to the 30 end of the viral genome (56). Genes that might down-regulate the expression of viral antigens (e.g., the M2-2 gene in RSV) can be deleted (57) and codon usage can be optimized to increase protein expression (58). In addition, the increased expression of IFN associated with the deletion of IFN antagonists can provide for increased immunogenicity. The sixth underlying principle in the development of live attenuated RSV and PIV vaccines (Table 4) is that phenotypic stability, that is, the maintenance of the att phenotype of a live attenuated virus following replication in vitro and in vivo, can be significantly enhanced using reverse genetics. Deletion mutations and hr phenotypes based on gene swapping are inherently refractory to reversion. While attenuating amino acid substitution mutations are inherently less stable, they can be replaced with amino acid deletions to increase phenotypic

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stability (59). Alternatively, stabilization can be achieved by selecting codons that require more than one nucleotide change to revert to wt phenotype (60). This sometimes involves the use of alternative amino acid assignments, which can provide for an increase in the level of temperature sensitivity and attenuation compared with the assignment in the original mutant (60–62). In addition, the stability of ts att substitution mutations can be increased by combination with non-ts att mutations, since the latter are not subject to the constant selective pressure of temperature. In addition, it often is possible to add additional att mutations without a proportional increase in attenuation, providing a virus in which an increased number of att mutations provides greater phenotypic stability.

Live Attenuated RSV Subgroup A Vaccines RSVcp248/404/1030DSH One of the most promising RSV subgroup A candidate vaccines is RSVcpts248/404/1030DSH, a vaccine virus that contains five att mutations or sets of att mutations and that has achieved an appropriate balance between attenuation and immunogenicity for young infants (19). RSVcpts248/404, the mutant virus on which RSVcpts248/404/1030DSH was built, contained a set of five non-ts att mutations from the RSVcp parent virus (49), a non-coding ts att mutation in the M2 GS sequence (the 404 mutation, a T to C nucleotide substitution at position 9 of the GS sequence), and a ts att amino acid substitution mutation in L (the 248 mutation, a Q831L change in L). RSVcpts248/404 virus was highly attenuated for seronegative older infants but retained the ability to cause nasal congestion for approximately one day that interfered with feeding and sleeping in young infants, precluding its use as a vaccine for this target age group (20). RSVcpts248/404 replicated to a mean peak titer of 104.0–4.2 PFU/mL in seronegative infants (20). To further attenuate RSVcpts248/404, the SH gene was deleted (DSH ) using reverse genetics. RSVcpts248/404DSH replicated in seronegative older infants as efficiently as RSVcpts248/404, and was not further developed as a vaccine candidate (19). Thus, the addition of the DSH mutation, a non-ts att mutation that attenuates RSV wt virus for chimpanzees, to RSVcpts248/404 did not result in further attenuation for seronegative subjects. However, the addition of the DSH mutation to RSVcpts248/404 likely would increase the overall phenotypic stability of RSVcpts248/ 404DSH, and therefore it was retained in subsequent constructs. The 1030 mutation, a ts att mutation in L (Y132N), was added to RSVcpts248/404DSH to create RSVcpts248/404/1030DSH, a mutant that exhibited a greater level of temperature sensitivity than RSVcpts248/404. This vaccine candidate therefore contained five att mutations, of which three were ts (404, 248, and 1030) and two were non-ts mutations (the cp mutations and the DSH mutation) that could each independently attenuate RSV for nonhuman primates or humans. Compared with RSVcpts248/404DSH, RSVcpts248/404/ 1030DSH is highly restricted in replication in RSV-seronegative children (mean peak titer 102.5 vs. 104.3 PFU/mL), indicating that the 1030 mutation has a strong attenuating effect on RSVcpts248/ 404DSH. In one- to two-month-old infants, 105.3 PFU of RSVcpts248/404/1030DSH, given intranasally, infected 94% of the vaccinees. Virus shedding lasted for approximately 10 to 14 days and the vaccine was well tolerated without the occurrence of nasal congestion characteristic of RSVcpts248/404 infection of young infants (19). The vaccine virus replicated to the same level in older seronegative infants as in one- to

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two-month-old infants, indicating that maternally derived RSV-specific antibodies did not significantly inhibit its replication in young infants. Vaccine virus shed in nasopharyngeal secretions retained the ts phenotype, and four of the five att mutations were retained in all isolates. However, a minority of isolates contained virus that exhibited a decreased level of temperature sensitivity due to a loss of one of the three ts mutations. These modified vaccine viruses did not become the predominant viral species in the vaccinees, but, rather, existed as a small subpopulation. Although not all infected vaccinees developed an immune response that was detectable by the available methods, the replication of a second dose of vaccine given four weeks after the first dose was restricted in the majority of vaccinees, indicating that some degree of protective immunity was induced (19). Whether the immune response is robust and durable enough to decrease LRTI caused by infection with wt RSV needs to be determined in expanded efficacy studies. Deletion of NS1, NS2, and M2-2 Four RSV gene deletion mutants containing deletions of SH, NS1, NS2, or M2-2 are viable and replicate efficiently in Vero cells, and thus they provide a new set of genetically stable candidate vaccines (Tables 4 and 5) (30). As noted, deletion mutants should be more stable than vaccine viruses whose attenuation is based solely on point mutations. Deletion of the nonstructural proteins NS1 and/or NS2 resulted in viruses that replicated almost as efficiently as wt RSV in Vero cell culture but exhibited significant attenuation in nonhuman primates. RSVDNS1, for example, was tenfold more restricted in replication in the URT of chimpanzees than RSVcpts248/404, the candidate vaccine that was only slightly under-attenuated in the one- to two-month-old infants (57). Whereas the DSH mutation reduced replication of wt RSV up to 80-fold in chimpanzees, the DNS1 deletion restricted replication over 20,000-fold, indicating that it has a profound attenuating effect

in nonhuman primates. RSV containing a deletion of DNS2 is less attenuated in chimpanzees than RSVDNS1 and RSVcpts248/404, indicating that it does not provide sufficient attenuation for use as a single-mutation vaccine. However, further attenuation probably can be achieved by including one or more att point mutations. Since RSVDNS1 is more attenuated than RSVcpts248/404 in chimpanzees but still induces protection against wt RSV challenge, it may be ideal for vaccination of infants. This virus has not entered clinical trials yet. In addition, since NS1 and NS2 antagonize the IFN type I-mediated antiviral state, deletion of NS1 or NS2 could potentially increase the immunogenicity and protective efficacy of an RSV vaccine (6,66–68). Deletion of M2-2, the second, downstream open reading frame (ORF) of the M2 mRNA, results in an attenuated virus with a level of attenuation and immunogenicity in chimpanzees similar to that of RSVDNS1. In RSVDM2-2 infected cells, the balance between transcription and RNA replication appears to be shifted toward increased transcription and decreased RNA replication. This provides a phenotype of increased antigen expression concomitant with restriction of replication that seems ideal for a vaccine since it theoretically would increase protein expression and thereby increase immunogenicity. Clinical evaluation of this virus is certainly warranted. In summary, gene deletion mutants represent a promising set of RSV candidate vaccines. Their level of attenuation in chimpanzees can be ranked and compared with RSVcpts248/404 as follows: DSH < DNS2 < RSVcpts248/404 < DNS1 < DM2-2 (57,69). Since RSVcpts248/404 is slightly under-attenuated for seronegative infants, RSVDNS1 or RSVDM2-2 are considered promising candidates for future clinical studies in young children. Bovine RSV BRSV contains hr att sequences and has been evaluated as a vaccine against human RSV (HRSV) (53,70). BRSV and HRSV

Table 5 Mutations or Natural Sequences Used to Attenuate RSV and PIV Viruses for Nonhuman Primates and Humans Type of mutation or genetic modification

Virus (example) F170S

1 Individual point mutation

rHPIV1-C

2 Serial passage at suboptimal temperature (cold passage) 3 Chemical mutagenesis (after cold passage) 4a Use of a related nonhuman virus

HPIV3cp45

RSVcpts248/404 BPIV3

5a Chimerization or substitution of individual viral genes 6a Combination of several point mutations 7a Codon deletion

B/HPIV3

8a Deletion of an entire viral gene 9a Codon modification

rRSVcp DNS2

rHPIV2-15C/ 948L/D1724 rHPIV1-CD170

rHPIV1-LY942A

Mechanism

Preclinical/clinical findings

References

Abolish IFN antagonist function of the HPIV1 C protein Combination of 5 mutations in several genes that confer att, ts and ca phenotypes Additive effect of multiple att mutations Multiple hr att genes; replicates well in bovines but poorly in humans Combine hr att with wild-type HPIV3 antigenicity

Attenuated and immunogenic in primates Safe and immunogenic in seronegative infants

63

Slightly under-attenuated in seronegative infants Safe but weakly immunogenic in seronegative infants

20

Additive effect of 3 individual att mutations Abolish IFN antagonist function of the HPIV1 C protein; more stable than substitution Abolish IFN antagonist function of the RSV NS2 protein Increase attenuation and genetic stability

46

24,52

Safe in adults

64 and Karron, unpublished

Attenuated and immunogenic in primates Attenuated and immunogenic in primates

61,65

Over-attenuated for adults; under-attenuated for infants Attenuated and immunogenic in primates

63

37 60

a Combines attenuation with increased genetic stability. Abbreviations: IFN, interferon; RSV, respiratory syncytial virus; PIV, parainfluenza virus; ca, cold adapted; hr, host range; ts, temperature sensitive.

Chapter 58: Vaccines Against RSV and PIV

share considerable antigenic cross-reactivity as measured with pooled human antibodies or HRSV F-specific monoclonal antibodies (71). BRSV itself has been considered as a live vaccine against HRSV, but BRSV strain A51908 did not replicate to a detectable level in seronegative chimpanzees and failed to induce resistance to challenge with HRSV, indicating that BRSV was over-attenuated in primates (70). To increase the replication of BRSV and to improve its immunogenicity against HRSV, cDNA-derived BRSV was modified so that the BRSV G and F genes were replaced by their HRSV subgroup A counterpart genes. Chimeric rBRSV/HRSV was still highly restricted in chimpanzees and did not induce significant protection against HRSV challenge (70). Replacement of additional BRSV genes in rBRSV/HRSV with their HRSV counterparts might improve replication of the chimeric virus in chimpanzees and achieve a satisfactory level of immunogenicity, but these studies in chimpanzees have not been conducted yet.

Live Attenuated RSV Virus Vaccines for Subgroup B A RSV vaccine should protect against both RSV subgroup A and subgroup B. Attempts to produce a satisfactorily attenuated subgroup B vaccine candidate by conventional methods have not been successful so far (72–74), and a reverse genetics system for subgroup B has not been used to develop attenuated RSV subgroup B mutants. However, it is possible that one or more of the cDNA-derived subgroup A vaccine viruses described above could be modified to contain the G and F glycoprotein genes of subgroup B in place of the subgroup A glycoprotein genes (75). Such RSVA/B chimeric vaccine viruses would be attenuated by the mutations in the RSV A backbone, but should induce antibodies to the G and F glycoproteins of RSV B. Initial studies in which a RSVA/B chimeric virus was generated from two wt RSV A and B mutants viruses showed that replication of the chimera in cell culture and in chimpanzees was comparable to replication of the RSV A and B wt viruses (75). This indicated that chimerization did not independently attenuate RSVA/B and suggested that mutations that satisfactorily attenuated RSV A vaccine virus could also attenuate the RSVA/B chimeric virus without causing over-attenuation. Since nearly all of the available att mutations identified for the RSV subgroup A strain lie outside the G and F genes, it is likely that the same att backbone can be used for the subgroup A and B vaccine viruses. For example, the RSVA/B chimeric virus that was constructed with the backbone of RSVcpts248/404/1030 (similar to the vaccine candidate described above except for the presence of the SH gene) possessed the same degree of temperature sensitivity as RSVcpts248/404/1030 and exhibited a similar degree of attenuation in chimpanzees (75). Thus, a live attenuated RSV vaccine could be created, which contained an attenuated A component and an RSVA/B chimeric virus with identical attenuating mutations. As a second approach to generating a subgroup B vaccine, the G protein of subgroup B could be inserted into the subgroup A genome as an additional gene unit (76). Since G is highly divergent between the subgroups whereas F is relatively well conserved, a single chimeric virus expressing the G proteins of both subgroups and the F protein of subgroup A alone could serve as a bivalent vaccine against both subgroups. Alternatively, a complete RSV subgroup B virus could be derived from cDNA and attenuated using the methodology described for RSV subgroup A above.

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Live Attenuated HPIV3 Virus Vaccines HPIV3cp45 HPIV3 is second only to RSV as a cause of viral bronchiolitis and pneumonia in infants and children (Table 1). Similar to the development of cpRSV, live attenuated candidate vaccines were developed by serial tissue culture passage of wt virus at suboptimal temperatures (Table 5). One of these candidate vaccines, HPIV3cp45, was cold-passaged 45 times and acquired the ca, ts, and att phenotypes in the process (38). Sequencing of HPIV3 wt and its HPIV3cp45 derivative identified 15 mutations in the HPIV3cp45 genome, and the contribution of the identified mutations to the three phenotypes was determined using reverse genetics (50,77). Five mutations, three ts mutations in L and two non-ts in P/C and F, independently contribute to the attenuation phenotype and provide a high degree of phenotypic stability of virus following replication in experimental animals and in humans. In clinical trials, HPIV3cp45 was found to be safe and well tolerated in all age groups, including infants as young as one month of age. Ten thousand 50% tissue culture infectious dose (TCID50) infected 94% of the one- to three-month-old seronegative infants, and vaccinees shed virus at a mean peak titer of 103.3 PFU/mL. All of the virus isolates tested retained the ts and ca phenotypes (48,78). Infants received a second dose of vaccine either one or three months after the first dose, and replication of the second dose of vaccine was restricted 40- to 100-fold, indicating that immunization resulted in the induction of protective immunity. Only a minority of the youngest infants developed a detectable HPIV3 specific IgG response, possibly due to the presence of maternal PIV3 specific IgG. However, HPIV3 HN-specific serum IgA, which is readily detectable in the presence of maternally derived IgG antibodies, was found to be a sensitive correlate of protection against replication of the second dose of vaccine (48). In a separate trial in 380 children 6 to 18 months of age that included 226 seronegative infants and children, HPIV3cp45 was also well tolerated, safe and immunogenic (46). Adverse events did not differ between vaccinees and placebo recipients, indicating that this vaccine virus had a satisfactory level of attenuation. Eighty-four percent of seronegative HPIV3cp45 vaccine recipients seroconverted to HPIV3, indicating satisfactory infectivity and immunogenicity (46). Compatibility between live attenuated RSV and HPIV3 components of an experimental bivalent vaccine was assessed by simultaneous intranasal vaccination with 105 PFU each of RSVcpts248/404 and HPIV3cp45 in 6- to 18-month-old seronegative children. In this trial, the infectivity of HPIV3cp45 was 76%, compared with 92% in the group given the monovalent vaccine suggesting that the high level of replication of RSVcpts248/404 might have interfered with the infectivity of HPIV3cp45. Nonetheless, the combined vaccination was immunogenic for both RSV and HPIV3, and antibody responses were not statistically different from that of the monovalent groups (79). It is possible that co-administration of a more attenuated RSV A or B component with HPIV3cp45 in a bi- or trivalent vaccine would result in less or no interference of the RSV component with the replication of HPIV3cp45. In currently ongoing clinical trials, cDNA-derived recombinant (r)HPIV3cp45 has replaced the biologically derived virus. Derivation of vaccine virus from cDNA adds to the safety profile of the vaccine because generation of virus from cDNA using Good Manufacturing Practices effectively bypasses the risk of possible viral or other biological contamination

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originating in the monkey kidney tissue used during the 45 passages of HPIV3cp45. Currently, rHPIV3cp45 is being evaluated in a phase 1 trial in seronegative infants 6 to 12 months of age to confirm the att phenotype of the cDNAderived virus and to add more safety data in this young age cohort. A phase 2 trial for this vaccine is in preparation as part of a Cooperative Research and Development Agreement between NIAID and MedImmune, Inc. B/HPIV3 and HPIV3-PB BPIV3 and HPIV3 are closely related viruses that (as with BRSV and HRSV) have separately evolved in their respective hosts. Their antigenic relatedness (approximately 25%) has been analyzed by reciprocal cross-neutralization (80), and studies in monkeys confirmed that the majority of the antigenic sites recognized by humans during HPIV3 infection were also recognized by sera of monkeys infected with BPIV3 (81). Replication of two different strains of BPIV3 was restricted 100- to 1000-fold in rhesus monkeys compared with HPIV3 (80), and infection of cotton rats and monkeys with BPIV3 induced resistance to subsequent challenge infection with HPIV3 (80). These findings, indicating that BPIV3 is restricted in replication in experimental animals compared with HPIV3 and that (unlike BRSV and HRSV) BPIV3 infection induced protection against HPIV3, formed the basis for evaluation of this virus in humans. In phase 1 trials, BPIV3 was found to be poorly infectious in adults and in children seropositive to HPIV3 (55,82). In seronegative infants and children, administration of either a 104 or 105 TCID50 dose of BPIV3 was found to be highly infectious, safe and immunogenic at either dose (24). Because of the antigenic differences between the BPIV3 and HPIV3 glycoproteins, the geometric mean HAI antibody titers in BPIV3 vaccinees were lower against HPIV3 than against BPIV3, and the seroconversion rate for HPIV3 was only 62% compared with 85% for BPIV3 (24). Subsequently, a placebo controlled phase 2 trial was conducted in 192 two-month-old infants with four doses of 105 TCID50 or 106 TCID50 administered at 2, 4, 6, and 12 to 15 months of age (52). BPIV3 was well tolerated and most adverse events were evenly distributed between vaccine and placebo recipients, but fever >388C was more common in vaccine recipients after the second dose of vaccine. Since viral cultures were not obtained in this study, the relationship between fever and level of vaccine virus replication could not be assessed. Again, seroconversion rates were satisfactory against BPIV3 but only modest against HPIV3 (52). To improve immunogenicity against HPIV3, a cDNAderived chimeric bovine/human PIV3 virus (rB/HPIV3) was constructed bearing the HPIV3 HN and F genes in place of the respective BPIV3 genes (64,83). In rhesus monkeys, rB/HPIV3 retained the att phenotype of its BPIV3 parent and induced higher titers of antibody against HPIV3 than did BPIV3 (64). Clinical studies of rB/HPIV3 have been initiated in adults and seropositive children. To understand the genetic basis of attenuation of BPIV3 for nonhuman primates, chimeric viruses were generated in which the N, P/C/D/V, M, or L gene of HPIV3 was replaced by its BPIV3 counterpart (54,84). These studies in rhesus monkeys indicated that each BPIV3 gene independently conferred some degree of attenuation. Thus, attenuation of BPIV3 for rhesus monkeys is polygenic. This finding supports previous observations that the BPIV3 att phenotype was stable

following replication in humans (55). Two of these bovine/ human PIV3 chimeric viruses, designated rHPIV3-NB and rHPIV3-PB, were as attenuated (rHPIV3-NB) or more attenuated (rHPIV3-PB) than BPIV3 in rhesus monkeys, and each retained immunogenicity and efficacy against HPIV3 (54). Phase 1 studies of rHPIV3-NB in HPIV3 seronegative children indicate that this virus is not as attenuated as HPIV3cp45 (Karron et. al., unpublished observations). Studies with rHPIV3-PB are planned.

Live Attenuated HPIV1 Virus Vaccines A number of strategies using reverse genetics have been explored to develop a live attenuated HPIV1 vaccine, including the use of HPIV3cp45 as a vector for HPIV1 antigens (85–87) or the introduction of attenuating mutations into full-length HPIV1 (59,60,63,88,89). Initially, the HN and F glycoproteins of rHPIV3cp45 were replaced with those of HPIV1 yielding a virus, designated rHPIV3-1cp45, that was as attenuated as HPIV3cp45 in hamsters and that offered protection against challenge with wt HPIV1 (86,87). However, rHPIV3-1cp45 exhibited decreased immunogenicity to HPIV1 in the presence of immunity to HPIV3, probably mediated by cellular immunity against the ‘‘internal’’ HPIV3 proteins. Since infants will likely be vaccinated against HPIV3 first and against HPIV1 later, an HPIV3-based HPIV1 vaccine such as rHPIV3-1cp45 might not infect and replicate efficiently in these infants and thus would be insufficiently immunogenic. To develop a non-chimeric HPIV1 vaccine, HPIV1 was generated from its own full-length cDNA (90) and att mutations identified in heterologous PIVs or RSVs were introduced into homologous sites of the HPIV1 genome. This strategy has proven to be successful, especially for mutations introduced into the P/C and L genes. The P/C gene of HPIV1 expresses multiple proteins from a single gene unit, including the phosphoprotein (P) and the four C-related proteins, C0 , C, Y1, and Y2, which are expressed from a single open reading frame that overlaps that of P (18). The C proteins of HPIV1 inhibit the production of type 1 IFN and signaling of IFN through its receptor (7). An att point mutation, CF170S, that was originally described in Sendai virus (a murine PIV1 virus) (91), was imported into HPIV1 to yield rHPIV1-CF170S. rHPIV1-CF170S was found to be attenuated for both the URT and LRT of hamsters and African green monkeys (AGMs) (7,63,88). This mutation affected each of the C proteins but was silent in the overlapping P gene. To increase the phenotypic stability of this mutant, a deletion of codon 170 in C (rHPIV1-CD170), was generated and was found to be as attenuated as rHPIV1-CF170S for nonhuman primates (7,63) (Table 5). rHPIV1-CD170 replicated efficiently in Vero cells despite a corresponding mutation in the overlapping P protein. A second rHPIV1 mutant with a mutation in C combined with a second, spontaneous mutation in HN, rHPIV1-CR84GHNT553A, was also attenuated for monkeys. Two mutations were imported from BPIV3 and HPIV3cp45 (L1711 and LY942H, respectively) into the HPIV1 L ORF. These mutations were stabilized by either codon modification (so that three nucleotide substitutions were required for reversion to wt—LY942) or by deletion of two codons (LD1710–1711) (59,89). rHPIV1s containing the LY942A and LD1710–1711 mutations were phenotypically stable in vitro and attenuated in AGMs. rHPIV1 vaccine candidates containing a combination of P/C gene and L gene mutations such as rHPIV1C R84G/D170 HN T553A L Y942A and rHPIV1-C R84G/D170 HN T553A

Chapter 58: Vaccines Against RSV and PIV

LD1710–1711 are highly restricted in replication in AGMs. Since the rHPIV1-CR84G/D170HNT553ALY942A vaccine candidate confers protection against HPIV1 challenge in AGMs, it will be evaluated in clinical trials (59).

Live Attenuated HPIV2 Virus Vaccines The development of a live attenuated HPIV2 vaccine, like that for HPIV1, is based on a cDNA-derived full-length HPIV2 (92,93). A number of HPIV2 mutants with ts and att phenotypes conferred by mutations in the L protein and in the 30 genomic promoter have been generated. Substitutions at amino acid positions 460, 948, and 1724 of the L protein—which were predicted from att mutations present in other viruses— generated mutants that were ts in vitro and att in hamsters and AGMs (65). Also, a spontaneous T to C nucleotide substitution at position 15 of the 30 genomic leader region specified a non-ts att phenotype (65). The non-ts 15T?C mutation restricted replication in both the URT and LRT of AGMs, whereas the substitution mutations in L conferred attenuation predominantly in the LRT, as would be expected for non-ts versus ts mutations (65). Two HPIV2 candidate vaccines with three att mutations each offer a desirable degree of attenuation and immunogenicity: rHPIV2(15C)/460A/948L and rHPIV2(15C)/ 948L/D1724 (65). Both viruses provided significant protection in AGMs against HPIV2 challenge, although the titers of antibody measured by a hemagglutination-inhibition (HAI) assay were only modest (65). Both viruses appear appropriate for clinical evaluation, and rHPIV2(15C)/948L/D1724 has entered into phase 1 trials in 2009.

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rhesus monkeys, rB/HPIV3-RSV chimeras were attenuated like their rB/HPIV3 parent, yet were highly immunogenic against both RSV and HPIV3 and protective against HPIV3 (this animal is not highly permissive to RSV and thus challenge was not performed) (96). These two rB/HPIV3-RSV chimeric viruses expressing the G and F proteins of RSV A and B from separate viruses are attractive candidates for use as a trivalent pediatric vaccine to protect against RSV A, RSV B, and HPIV3. The att phenotype conferred by the BPIV3 backbone is very stable, and co-immunization against RSV and PIV3 is desirable since the two viruses are similar in their epidemiology and the target population is the same. MedImmune has initiated phase 1 trials in adults and seropositive children with a rB/ HPIV3-RSV chimera that expresses the F protein of RSV subgroup A from an additional gene unit inserted between the N and P gene of B/HPIV3 (97,98). This bivalent vaccine against HPIV3 and RSV was found to be safe and well tolerated in adults (39), but data from studies in children have not been published yet. PIV vectors expressing RSV antigens have important advantages compared with wt RSV and attenuated RSV strains: specifically, they replicate more efficiently in vitro (providing for more efficient manufacture) and are physically more stable (providing for easier manufacture, distribution, and use). PIV1 can also be engineered to express the protective antigens of other paramyxoviruses. It should be possible to develop a PIV1 vector with HPIV2 HN and F glycoproteins expressed from additional gene units containing, if needed, one or more of the stabilized mutations indicated above.

OUTLOOK AND CONCLUSION Vectored RSV and PIV Vaccines Several non-paramyxovirus vectors expressing RSV or PIV glycoproteins have been evaluated in experimental animals, including replication competent and replication defective vector viruses. The major advantage of using a vector to express RSV or PIV protective antigens, as opposed to a subunit protein vaccine, is that the RSV or PIV antigen is expressed by a human cell in a native conformation comparable to that expressed in response to RSV or PIV infection. Since the nonparamyxovirus vectors expressing RSV or PIV antigens have not progressed into clinical trials, they will not be further described in this chapter and the reader is referred to recent reviews that discuss the properties of some of these vectors in more detail (30,94). The preferred strategy with paramyxovirus vectors has been to take one virus for which a vaccine is needed and to use it as a vector to accept an additional gene expressing the protective antigens of a second virus for which a vaccine is needed, thus creating a bivalent vaccine virus. One such vectored vaccine has already entered clinical trials, as described below. At present, the most promising vector for RSV and PIV antigens is the previously described rB/HPIV3 chimeric virus expressing RSV F or G proteins. This chimeric virus has the HN and F proteins from HPIV3 to induce resistance to HPIV3, the BPIV3 hr restriction for attenuation in primates, and an RSV G or F gene insert to induce resistance to RSV. Thus, one virus can protect against both RSV and PIV3. Two such recombinant viruses expressing the F and G from RSV subgroup A or from RSV B were generated (95–97). The RSV G or F ORFs were inserted into the promoter-proximal position of rB/HPIV3 to achieve a high level of expression and immunogenicity. In

Fifty years of vaccine development have not yielded a licensed vaccine against RSV or PIV, but we are much closer to achieving this goal than ever before. The ability to create a menu of mutations that can incrementally attenuate RSV and PIVs in vivo without a significant effect on virus growth in vitro should enable the development of RSV and PIV vaccines that possess an acceptable balance between attenuation and immunogenicity in the respective target populations. The present menu of mutations, including non-ts point mutations, ts point mutations, codon deletions, gene deletions, and hr determinants, has generated a number of promising cDNA-derived candidate vaccines that need to be evaluated in clinical studies. For RSV, candidate vaccines that include deletion of the NS1 or M2-2 gene seem particularly promising because they might be able to induce protective immunity in spite of their high level of attenuation. Although this overview focused on pediatric vaccine development, it should not be overlooked that RSV is also a significant cause of respiratory tract disease in elderly populations (99,100). A vaccine virus that is appropriately attenuated for young infants will be over-attenuated for adults, and it will be important to define a second group of candidate vaccines that are appropriate for evaluation in the elderly. Perhaps non-paramyxovirus vectors or subunit RSV vaccines also might have usefulness in this population. For HPIV3, rHPIV3cp45 or rB/HPIV3-based vaccines are promising. Efficacy trials for rHPIV3cp45 will take several years to complete, however. For HPIV1 and HPIV2, preclinical data indicate that several live attenuated mutants are good candidates for clinical trials. It is anticipated that vaccines against RSV and PIV will significantly decrease hospitalization for pediatric respiratory tract disease in the not too distant future.

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ACKNOWLEDGMENT This research was supported by the NIAID Division of Intramural Research.

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59 Cytomegalovirus Vaccines David I. Bernstein Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio, U.S.A.

Stanley A. Plotkin Wistar Institute of Anatomy and Biology, University of Pennsylvania, Doylestown, Pennsylvania, U.S.A.

INTRODUCTION Human cytomegalovirus (HCMV) infection causes a spectrum of diseases in children and adults. In healthy individuals, it can cause a mononucleosis-like disease or more commonly, an asymptomatic or unrecognized infection. In immunocompromised patients, especially those with advanced AIDS or solid organ and hematopoetic cell transplants, it is a common cause of serious illness and can be a fatal infection. More recently, published data have suggested a possible role of HCMV in atherosclerosis (1), immunosenescence (2) and autoimmune disease (3). HCMV is also the cause of the most prevalent maternal infection that is transmitted to the fetus. This congenital infection may produce severe, mild, or no disease at birth, and may only manifest itself later in life (4,5). Prevention of congenital disease is the main incentive for developing HCMV vaccines. In general, postnatal HCMV infection is quite common throughout the world, with seroprevalence rates of 50% to 90%. Seroprevalence is inversely related to socioeconomic level but directly related to the intensity of contact between toddlers, who acquire HCMV in day care. The availability of antivirals and intravenous immunoglobulins has led to a decreased incidence of HCMV diseases in some high risk populations, but those modalities provide incomplete protection. Recently, an observational study demonstrated that administration of cytomegalovirus (CMV) hyperimmune globulin to pregnant women with a primary CMV infection during pregnancy profoundly reduced sequelae in the infants (6). Confirmation of these findings could affect our screening strategies for congenital CMV and treatment. However, given the limited treatment and prevention options available today, an effective vaccine against HCMV would be highly desirable.

CLINICAL MANIFESTATIONS Congenital Infection Severe congenital infections with HCMV were first recognized in the early 1900s when they were termed cytomegalic inclusion disease (CID), because of the nuclear inclusions seen on histologic examination. HCMV is the most common congenital infection with 0.2% to 2.5% of newborns infected in utero (&40,000 infants in the United States) and the most common infectious cause of congenital abnormalities (5). Severe disease is seen in about 10% of congenitally infected infants after primary infection and is

characterized by intrauterine growth retardation, hepatosplenomegaly, jaundice, a rash often described as ‘‘blueberry muffin’’ like, and severe central nervous system (CNS) involvement including microcephaly, intracranial calcifications, and chorioretinitis. Laboratory abnormalities include thrombocytopenia, elevated liver function levels, and hemolytic anemia. Most infants severely affected at birth develop significant neurologic sequelae with a mortality of 4% to 37%. The risk of sequelae in symptomatic and asymptomatic infants has recently been correlated directly to the viral load in the infants (7). The most common sequelae of congenital HCMV are progressive sensorineural hearing disease, which can be seen in both, infants who are symptomatic or asymptomatic at birth (8,9). The risk of developing hearing loss has recently been correlated with the detection of viremia in children with symptomatic congenital CMV involving the CNS (10), and with the amount of virus in the blood and urine in those who were asymptomatic at birth (11). The precise number of affected infants born annually in the United States is unknown, but the estimate by Fowler et al. is about 8000 infants (12). Cost-benefit analyses have suggested that an estimated $834 million would be saved annually by immunization with a moderately priced vaccine (13), assuming complete immunization of all women aged 15 to 24 years. Important distinctions have been drawn between congenital infection, which follows a primary maternal infection and that which results from an infection in a previously infected mother (12,14–20). Transmission to the fetus occurs in about 30% to 50% of pregnancies complicated by a primary HCMV infection during pregnancy compared to about 1% following recurrent infection. Initial reports suggested that severe sequelae were almost exclusively seen following pregnancies complicated by primary HCMV infection (9,15). For example, Fowler et al. (12) found that sequelae occurred in 25% of 125 infants with congenital infection following primary CMV infection during pregnancy but in only 8% of 64 infants with congenital infection born to mothers who were HCMV seropositive prior to pregnancy. More importantly, none of the infected infants born to seropositive mothers developed severe sequelae; defined as bilateral hearing loss or mental retardation (an IQ < 70). These sequelae occurred only among infants born to mothers who had a primary infection during pregnancy. Such observations suggest that maternal immunity to HCMV prior to pregnancy prevents the majority of severe sequelae

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associated with congenital infection and form an important argument for the feasibility of developing HCMV vaccines to prevent congenital HCMV disease. Other studies (18–24) suggest that symptomatic congenital infection and permanent neurologic sequelae are not as rare in infants of women with preconceptional immunity as previously thought. Clearly symptomatic congenital infection can occur after nonprimary or recurrent maternal infections, but the extent of the risk is not clear. In a large study of congenital infection, a previous infection provided a 69% reduction in the risk of congenital CMV infection compared to women with a primary infection during pregnancy (23). Determining whether infections in mothers with preexisting immunity are due to reactivated virus or reinfections has significant implications for the development of vaccines. Recent evidence, based on the antibody response to specific variable epitopes on the amino terminal region of glycoprotein H, suggests that reinfection may account for a substantial portion of symptomatic congenitally infected infants born to women with preconceptional immunity (25). Recently, a study performed in Brazil substantiated a 1% incidence of congenital CMV infection in a highly seropositive population, with fetal abnormalities in at least 8% of infected infants (26).

HCMV Infections in Immunocompromised Individuals Bone marrow and solid organ transplant patients are both at risk to develop HCMV disease (27–31). Depending on the serostatus of the donor and recipient and the level of immunosuppression, the onset of most active HCMV infections is from two weeks until several months after transplantation. The mortality rate remains at about 5% even with available therapies. Besides the direct effects of infection, HCMV infection also increases immunosupression, elevating the risk for fungal and other infections, and increases the risk for organ rejection and/ or organ dysfunction (30). The risk of HCMV disease is least in kidney transplants, followed by heart and liver transplants (8–35%), while the highest risk is in pancreas (50%) and lung or lung-heart transplants (50–80%) (28). The most common clinical manifestations include fever, pneumonitis, hepatitis, leukopenia, gastroenteritis, and encephalitis. Bone marrow transplant recipients who receive bone marrow from a seropositive donor are at risk of developing HCMV disease from the virus that may be transmitted from the donor regardless of their own CMV serostatus because they have lost their own CMV immunity. In contrast, among patients receiving solid organ transplants, the risk of developing HCMV disease is highest among those who are seronegative prior to transplant and receive an organ from a seropositive donor. HCMV disease, however, also occurs among recipients who are seropositive prior to transplant. In such patients, HCMV infection may occur as a result of reactivation of the patient’s own virus or reinfection with HCMV strains introduced via the transplanted organ. Recent improvements in diagnostic assays including polymerase chain reaction, and antigen (pp65) detection, and the availability of prophylactic (ganciclovir, acyclovir, valganciclovir, and hyperimmune HCMV serum) and therapeutic drugs (ganciclovir, valganciclovir, cidofovir, and foscarnet) have dramatically changed the way HCMV infections are approached in transplant recipients (29,32). However, resistant viruses are a growing problem (33,34). HCMV is also an important cause of opportunistic infection in HIV-infected individuals (35–37). HCMV retinitis is the most common disease manifestation in this population, but

gastrointestinal manifestations including oral ulcers, esophagitis, colitis, cholecystitis, pneumonia, and CNS involvement are also seen. The advent of highly active antiretroviral therapy (HAART) has decreased the incidence of HCMV disease by decreasing the profound immunosuppression that leads to HCMV reactivation and disease, but continued attention to this important pathogen is required (36,38). Interestingly, in some patients, restoration of the immune response to CMV has resulted in immune-mediated disease manifestations (39).

MICROBIOLOGY CMVs are the principle members of the betaherpesvirus subgroup of the Herpesviridae family. CMV is an enveloped virus with a large icosahedral capsid surrounded by a tegument. The double-stranded DNA genome of HCMV is approximately 235 kb in size. The unique long sequence, UL, and the unique short sequence, US, of HCMV are flanked by repetitive sequences that are inverted relative to each other allowing for the presence of four isomeric forms. The replication of CMV is slow, at least 24 hours, and replication is highly species specific. In vitro, human fibroblasts show the greatest susceptibility to infection while in vivo fibroblasts, epithelial cells, macrophages, smooth muscle, and endothelial cells can support replication (40). The macrophage is the probable major site of latency, together with other cells of myeloid derivation (41–43). The purified virus contains 30 to 40 polypeptides including seven capsid proteins, perhaps as many as 60 glycoproteins (8 major), and 25 proteins making up the tegument or matrix (40,44,45). The most prominent glycoproteins are found as complexes. Glycoprotein complex I (gcI) antigen, glycoprotein B (gB), is encoded by UL 55. This major envelope glycoprotein appears as a heterodimer of two cleaved products, 93 and 55 kd in size (44,45). It plays a critical role in virus entry, and is the most important target for neutralizing antibody. The HCMV glycoprotein complex II (gcII) includes gM (UL 100), the most abundant glycoprotein (46) and gN (UL73). The gcIII complex consists of gH, gL, and gO, UL75, UL115, and UL 74, respectively. These proteins play an important role in virus entry. Recently four distinct gB, two distinct gH, and seven gN genotypes have been observed, which are useful in epidemiologic studies (25,47–53). The implications of this heterogeneity for vaccine development is unclear, but has raised concerns about the utility of a single glycoprotein vaccine such as gB. An important finding in recent years has been that entry into epithelial and endothelial cells is poorly prevented by neutralizing antibodies to gB, but is prevented by antibodies to a complex of proteins involving glycoproteins gH and gL plus the proteins produced by the UL128-131 genes located in the ULb’ region of the genome, which is lost with cell culture passage of the virus (54,55). Thus, neutralization in vivo may require multiple antibodies.

IMMUNOLOGY Protection from CMV infections is multifactorial. Innate, humoral and cell-mediated immunity all contribute to protection. Initial studies suggesting a role for innate immunity came from mouse experiments showing that animals deficient in functional natural killer (NK) cells were more susceptible to CMV. Later, Cmv-1, the gene that controls the initial splenic replication of murine CMV (MCMV) was linked to NK cell activity (56) and the NK cell subset and receptor, Klra8, that confers susceptibility was identified (57–59). NK cells may also be an important link to the adaptive immune response by supplying interferon gamma (IFN-g) and other cytokines.

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Evidence linking protection to antibody comes from several sources. Mouse studies have found that passive transfer of antibody can provide protection from a lethal infection (60,61). Further, using the guinea pig model of congenital infection, two groups have shown that passive transfer of antibody can protect the fetus from a lethal infection (62,63). Further support for a role of antibody in protection comes from neonatal transfusion studies (64,65). These studies showed that premature newborns born of HCMV seronegative mothers developed symptomatic CMV infections following transfusion with blood carrying the virus, but that the premature newborns born to HCMV seropositive mothers remained asymptomatic after receiving the same blood products, indicating that maternal antibody modulates HCMV infection. Most recently, an observational study demonstrated that CMV hyperimmune globulin administered to pregnant women with a primary CMV infection during pregnancy reduced disease in the infants (6). These findings suggest that antibody can prevent the initial acquisition of infection or the spread of the initial infection in the placenta and the fetus, in distinction to ongoing control of the virus, which clearly requires T cells. The role of antibody in protection following organ transplantation is less clear (66), except for kidney transplants where passive immunization does not protect against infection but decreases the severity of disease in seronegative recipients of kidneys from seropositive donors (67). The proteins that appear to induce the most consistent antibody response include glycoproteins gB(UL55), and gH (UL75), the tegument proteins pp150 (UL32), the matrix protein pp65 (UL83), and the nonstructural DNA binding phosphoprotein pp52 (UL44) (68). Neutralizing antibody is induced primarily by gB, 60% to 70%, but also to gH (69–71) and gM (72,73). The critical role of cell-mediated immunity and protection from CMV is documented by animal and human studies. Following CMV infections, the number of CMV-specific CD 8þ T cells increases to extraordinary high levels, and although the numbers fall rapidly, the number of circulating T cells that recognize CMV proteins remains high compared to most pathogens. Perhaps the best evidence is the fact that depletion of T-cell responses following HIV infections or transplants leads to severe HCMV disease. Early mouse studies demonstrated that suppression of T-cell function led to reactivation and dissemination of MCMV (74,75), while adoptive transfer studies confirmed a role for CD8þ cytotoxic T lymphocyte (CTL) in protection (76,77). Similarly, in transplant patients, a correlation between recovery of the cytotoxic T-cell response and recovery from HCMV infections has been identified (78,79), while adoptive transfer of CD8þ cytotoxic T clones were able to reconstitute the cellular immune response to HCMV and possibly provide protection (80). A role for CD4þ T cells has also been proposed (81–83), including a role in the protection of the fetus from congenital infection (84). In humans, several targets for the CD8þ CTL response to HCMV have been identified. The predominant target is p65 (UL83), although responses to IE1 (UL123), pp150 (UL32), and gB (UL55) are also frequent (85–87). Most recently, 151 ORFs were found to be recognized by CD4þ and/or CD8þ T cells (88), demonstrating the diversity of T-cell targets that are available.

ANIMAL MODELS The lack of an immune competent animal model in which HCMV replicates has hindered development of a HCMV vaccine. Thus, most animal models utilize species-specific

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CMV strains that are similar but not identical to HCMV. More recently, HCMV has been utilized in severe combined immunodeficiency (SCID) and nude mouse models (89,90), but thus far these have little relevance to vaccine evaluation. Animal models of CMV include mouse, guinea pig, rat, and rhesus monkey (91). Murine CMV models have provided important insights into the immunology and pathogenesis of CMV, but differences in placental structure severely limit their use for evaluating congenital CMV. Primate models are of interest for studying congenital CMV, but the cost and ubiquity of natural simian CMV infection makes these studies difficult (92). However, the rhesus model has been improved recently with the development of CMV-free monkey colonies. These have enabled pathogenesis and vaccination studies to be performed, which have demonstrated on the one hand that seropositive animals can be superinfected, but also that protection against challenge could be demonstrated (93,94). Nevertheless, the guinea pig model has become the model of choice for initial investigation of vaccines for congenital infection because the placental structure of the guinea pig is similar to that of humans (95), and the cost of study is reasonable. Guinea pig CMV (gpCMV) infection of the pregnant guinea pig produces a maternal infection that can be lethal or cross the placenta to induce fetal resorption, fetal death, or fetal growth retardation, depending on the challenge dose and time of virus challenge in relation to pregnancy (96). Primary infection of the pregnant animal typically produces a vertical transmission rate of 40% to 80%, similar to that in women following a primary HCMV infection. Several groups have used this model to explore protection from live virus, killed and subunit vaccines (91,97–99). This model has also been used to examine the protective effect of gpCMV antibody alone, because it is not clear if a CMV vaccine will need to induce cell-mediated immune responses or whether antibody alone would provide protection by neutralization or other mechanisms. In two studies, passive transfer of high titer antibody provided significant protection against pup mortality but results on protection from infection differed (62,63). Thus, these studies predicted the successful use of passive antibody in human trials of congenital CMV (6), and further suggested that antibody alone may provide protection against congenital disease. Vaccine evaluations using the guinea pig model of congenital infection provide evidence for the utility of a subunit gB vaccine (98–100) (Table 1) as well as the T-cell target, pp65 (91). Vaccination decreased pup mortality, shortened maternal viremia, and decreased infection rates in pups. Thus, this animal model also predicts that the gB vaccine currently in human trials will be effective, although it is not clear if addition of other proteins that induce neutralizing antibody such as gM/ gN (101) or those more likely to induce CTLs such as pp65 or IE1 would provide additional benefit. These strategies, however, can be evaluated in this model.

VACCINES The development of a CMV vaccine is a major priority. In fact, when the Institute of Medicine reviewed the priorities for vaccine development, a vaccine to prevent CMV was given the highest priority based on the economic cost savings (102). The main public health objective of a HCMV vaccine is to prevent symptomatic congenital HCMV disease. This can be accomplished by preventing HCMV infection of pregnant women, by modifying the infection so that virus is not passed

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Table 1 Effect of Immunization With gpCMV gB on Pup Mortality Following gpCMV Challenge of Pregnant Guinea Pigs Litters Group Control (unvaccinated) gB vaccine With Freund’s adjuvant With alum

Pups

Total

With  1 dead pup N (%)

Total N (%)

14 26 12 14

12 (86) 13 (50)a 4 (25) 9 (64)

41 91 42 49

Dead N (%) 31 (76) 23 (25)b 6 (14)c 17 (35)

p < 0.05 versus control group. p < 0.00001 versus control group. c p < 0.05 versus the group immunized with gB and alum. Abbreviations: CMV, cytomegalovirus; gB, glycoprotein B. Source: From Ref. 100. a b

to the fetus, or by modifying the fetal infection so that it does not induce disease. A secondary objective is to provide HCMV immunity and limit the number of HCMV infected individuals prior to a time when they might become immunosuppressed due to transplantation, HIV infection, or for other reasons. CMV vaccines may also be used in the transplant setting to increase the immunity in the donors of hematopoietic stem cells or the recipient of the solid organ transplants. The consequences of congenital HCMV and thus the burden of this disease on society are at least equal to other pathogens for which vaccines are available, including rubella, H. flu meningitis, and pneumococcal meningitis. Yet this need, although recognized for many years, has not been met (103). Delays in vaccine development are due to the difficulty and cost of evaluating HCMV vaccine efficacy, the uncertainty regarding the correlate(s) of protection, and the concern regarding persistence or possible reactivation of live virus vaccine, as well as the lack of priority given to HCMV vaccines by vaccine companies. Approaches to HCMV vaccines have included live attenuated vaccines, subunit and killed vaccines, DNA vaccines, and vectored vaccines.

Subunit Vaccines Although the ideal candidates for a subunit HCMV have yet to be defined, much of the interest has centered on gB because gB (UL55) is the major target of neutralizing antibodies (69,104–106). Antibodies to gB are thought to represent 60% to 70% of the HCMV-specific neutralizing antibody response (69), with the majority of this response directed to the AD1 region of the protein (residues 560–635), also known as the principal neutralizing domain (105). In addition to neutralizing antibody response, several laboratories have reported that gB can sometimes elicit MHC-restricted, CD8þ cytotoxic lymphocyte responses and proliferative CD4þ T-cell responses following natural infection (107). Recent reports of the heterogenicity of gB and gH (25,47–52), especially as it pertains to congenital infection (25,52), have raised concerns about the utility of a single protein vaccine. Additional Table 2 Leading Targets for Subunit CMV Vaccines Glycoproteins gCI (gB, [UL55]) gCII ( gM, [UL100], gN [UL73]) gC III ( gH [UL75], gI [UL115], gO, UL74) Other structural proteins pp65 (UL83) Nonstructural proteins IE1 (UL123)

Major inducer of neutralizing antibody Induce neutralizing antibody Induce neutralizing antibody

CTL target CTL target

Abbreviations: CMV, Cytomegalovirus; CTL, cytotoxic T lymphocyte.

proteins of interest for a subunit vaccine would include other glycoprotiens that induce neutralizing antibody such as the gcII and gcIII complexes, and those that are the major targets for cytotoxic T cells, pp65 and IE1 (Table 2). Sub unit vaccines may be developed as purified proteins, usually administered with an adjuvant, DNA vaccines, and vectored vaccines. Recombinant Glycoprotein B Vaccine Two large adult human trials and one smaller trial in toddlers evaluating a subunit gB vaccine developed by Chiron Corporation (Emeryville, California, U.S.) and combined with an oil-inwater based adjuvant, MF59, have been reported (108–110). The HCMV gB vaccine was derived from Chinese hamster ovary cells that were stably transfected with the coding sequence of HCMV Towne strain gB, from which the membrane spanning domain was deleted to facilitate secretion of HCMV gB, and the single protease cleavage site was mutagenized (111) to produce an uncleaved protein of 807 aa in length with 19 putative Nlinked glycosylation sites. The adjuvant MF59 is a squalene-inwater emulsion that induces higher gB antibody titers than does alum (108,109), the only currently approved adjuvant for human vaccines in the United States. MF59 has been used in several other vaccine candidates, including herpes subunit glycoprotein D herpes simplex virus vaccine (112) and avian influenza vaccines (113,114). It is also approved as an adjuvant for seasonal influenza vaccines in several countries in Europe. In both trials, the gB glycoprotein in doses ranging from 5 to 100 mg, combined with 10.75 mg of MF59 was shown to be safe and immunogenic. Evaluations included vaccine regimens of zero, one, and two months; zero, one, and four months; and zero, one, and six months, and a comparison of alum versus MF59. Immunization at zero, one, and six months induced the highest level of antibody with neutralizing and gB ELISA antibody titers that were higher than after natural infection (108,109). An optimal HCMV gB dose between 5 and 30 mg was determined, and groups that received MF59 developed higher titers compared with alum gB recipients (Fig. 1). The study in toddlers, 12 to 35 months of age, revealed that a dose of 20 mg plus MF59 was safe, and induced higher antibody titers than found in immunized adults (110). One of these trials has been completed and published and represents the most significant advancement in CMV vaccine in a long time. The first efficacy trial with this vaccine has been published recently (115). Three doses of vaccine or placebo were administered to 441 CMV seronegative women within 1 year after they had given birth. The vaccine appeared to be safe but there was an increase incidence of local reactions and some mild systemic reactions in the vaccine group. Although halted after an interim analysis indicated benefit, there were 49 confirmed infections, 18 in the vaccine group and 31 in the

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Figure 1 Anti CMV gB response in subjects immunized at zero, one, and six months with a subunit gB vaccine. The anti gB response of CMV infected individuals is provided for comparison. Abbreviations: CMV, Cytomegalovirus; gB, glycoprotein B. Source: From Ref. 108.

placebo group. Kaplan-Meier analysis revealed a significant increase in the number of vaccine recipients that remained uninfected during follow-up. Vaccine efficacy was 50% (95% confidence interval, 7 to 73) on the basis of infection rates per 100 person-years. One congenital infection among infants of the subjects occurred in the vaccine group, and three infections occurred in the placebo group. In another multicentered trial sponsored by the NIH, young, 12- to 18-year-old CMV seronegative females, who are at high risk of acquiring a CMV infection (116), are being vaccinated with MF59 or placebo on the same schedule and are also being evaluated for evidence of CMV infection by periodic antibody, polymerase chain reaction (PCR), and viral isolation. In a third trial, the gB vaccine is being evaluated for protection after solid organ transplants. The use of plant expression systems has paved the way for delivery of oral vaccines through edible transgenic plants. Recently, a group expressed the HCMVgB in tobacco plants as a model system (117). They found that transformed plants produced antigenic gB at levels of 70 to 146 mg/mL extracted protein. Peptide Vaccines The identification of immunodominant CMV epitopes has led to the development of peptide vaccines (118). Because of the probable importance of CTL induction to protection from CMV disease, CMV peptide vaccination with CTL epitopes of pp65 is being explored (119,120). Lipid modification at the amino terminal of one peptide produced a vaccine that did not require the use of adjuvant (119). More recently, using a new approach, positional scanning synthetic combinatorial libraries, researchers were able to modify the human leukocyte antigen (HLA)-A* 0201 pp65 (495–503) epitope to enhance its immunogenicity (120). The fusion of a promiscuous tetanus Th epitope to the pp65 CTL epitope produced an even more robust response. Although peptide vaccines are limited by the requirement for HLA allele-specific peptide motifs, estimates suggest that epitopes from two CMV proteins would be sufficient, even for diverse, multiethnic populations (121). Vectored Subunit Vaccines In this approach, the gene product of interest is expressed by an attenuated viral or other vector. In this way, the target protein

is expressed within a cell and thus is presented to the immune system by the class I pathway to efficiently induce cellmediated immunity in addition to humoral immunity. Canarypox recombinants. Canarypox virus is considered to be a good candidate vector for recombinant vaccines. Canarypox can accommodate large amounts of foreign DNA, which can be expressed during abortive replication in infected mammalian cells (122). Canarypox and other fowlpox viruses productively infect avian cells but do not produce infectious virus in mammalian cells, providing a safety factor not present for vaccinia virus. Canarypox infection of mammalian cells results in transcription and translation of early genes including genes that are inserted downstream of early promoters, thus inducing both humoral and cell-mediated immune response to the inserted gene product. Recombinant canarypox expressing the glycoprotein B of HCMV Towne strain was initially evaluated in mice and guinea pigs. Immunization appeared to be safe, and induced neutralizing antibodies and CD8þ CTL responses (123). Vaccination of HCMV seronegative humans with this vaccine, however, failed to induce significant gB ELISA or neutralizing antibody (124). In later experiments, this gB expressing vector was shown to prime for the induction of a booster response following subsequent Towne vaccination, as discussed below (124). Because one of the main advantages to vectored vaccines is the induction of CTLs, there is still interest in using canarypox as a vector for expressing major CTL targets such as pp65. Indeed, recent evaluations of canarypox expressing pp65 showed that it induced HCMV-specific CD8þ CTL, helper T lymphocyte, and antibodies (125). CTLs were elicited after only two vaccinations, and were still detectable 12 and 36 months after vaccination. Other vectors. Several vectors, including adenovirus and vaccinia, have also been used to deliver CMV genes (126–131). A recombinant adenovirus virus expressing gB was found to be immunogenic when given intranasally (127,130), and vaccinia expressing the dominant CTL target of mice, pp89, protected animals from a lethal challenge through a CD8þ T lymphocyte response (128). Using synthetic peptides spanning the IE1 epitope of pp89, also expressed in vaccinia, other investigators were also able to elicit protective CD8þ T-cell responses (132). Using the modified vaccinia virus Ankara (MVA) to express a

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soluble form of CMV gB, Wang et al. (129,133) demonstrated induction of high titer CMV neutralizing antibody in both naive mice and those with preexisting vaccinia immunity. More recently, Rist et al. (131) created a fused gene coding CTL epitopes from eight different CMV antigens that are accepted by 16 HLA class I alleles that was incorporated into a replication-deficient adenovirus 35 vector for delivery. Ex vivo stimulation of human peripheral blood mononuclear cells (PBMCs) with this vector stimulated multiple epitope-specific T cells. Replicon vaccines. Several alphavirus replicons have been evaluated as potential vaccine vector systems for infectious diseases and cancer (134). Use of virus-like replicon particles (VRP) based on Venezuelan equine encephalitis (VEE) virus is especially attractive because they express heterologous proteins to high levels, target expression to dendritic cells, and are capable of inducing both humoral and cellular immune responses to the vectored gene products. The VEE virus structural protein gene region can be deleted and replaced by a foreign gene in a cDNA plasmid (Fig. 2). An RNA transcript from such a plasmid, when introduced into cells, will be amplified and will express the foreign gene. This selfamplifying RNA (replicon) will direct the synthesis of very large amounts of the foreign gene product within the cell, typically reaching levels of 15% to 20% of total cell protein (135). Replicon RNA is packaged into VRP when cells are cotransfected with replicon RNA and two separate helper RNAs, which together encode the full complement of VEE virus structural proteins (135). Because the replicon RNA lacks critical portions of the VEE virus genome (i.e., the VEE virus structural protein genes) necessary to produce virus particles, VRP are propagation defective. Thus, VRP can infect target cells in vitro and in vivo and express the foreign gene to high levels, but are genetically restricted to a single cycle of replication. Upon immunization, VRP mediate the introduction of the replicon RNA into host cells, leading to high-level expression of

Figure 2 VRP vaccine packaging system. A gene encoding a foreign protein is substituted for the VEE structural genes to form the replicon RNA. The replicon and helper component (capsid protein and envelope glycoprotein RNA) are then cotransfected into cells and the replicon RNA packaged into VRP. Because the replicon RNA lacks critical portions of the VEE virus genome, VRPs cannot produce infectious particles and are replication defective but do express the foreign gene at high levels. Abbreviations: VRP, virus-like replicon particle; VEE, Venezuelan equine encephalitis.

antigenic proteins. VRP have been shown to specifically target antigen-presenting dendritic cells in vivo and are capable of inducing a broad array of immune responses to the foreign gene product, including CTL lymphoproliferative responses and neutralizing antibodies. Moreover, VRP have been shown to confer protection in animal models against a variety of diseases that require humoral and/or cellular effector mechanisms for protection (134). Several CMV proteins have been expressed in replicons including pp65, IE1 and gB, and were shown to be immunogenic in mice (136). The proof of principal experiment demonstrating that vaccination with the VEE replicon expressing the gpCMV homolog of pp65 could protect against congenital infection was recently published (91). Vaccination of guinea pigs prior to a pregnancy decreased the maternal viral load after gpCMV challenge, and decreased pup mortality in delivered pups in vaccinated dams (13%) compared to those immunized with a non-CMV encoding replicon (57%). A two component alphavirus replicon particle vaccine expressing CMV gB or a pp65/IE1 fusion protein (AlphaVax Inc., Research Triangle Park, North Carolina, U.S.), has recently been evaluated in a Phase 1 clinical trial in CMV seronegative adults (137). The vaccine appeared to be safe and induced ex vivo, direct IFN-g ELISPOT responses to CMV antigens and neutralizing antibodies in all subjects. Further, polyfunctional CD4+ and CD8+ T cell responses were detected by polychromatic flow cytometry.

Live Attenuated Towne The Towne strain vaccine was developed in the mid-1970s by multiple passages of an isolate from a congenitally infected infant in human fibroblast cells. Towne vaccine remains the most widely evaluated HCMV vaccine (138). The first clinical trial utilized pass 125 (MRC-5 cells) in healthy male subjects (139). Infection was not seen following intranasal administration, but following subcutaneous inoculation, all seronegative volunteers seroconverted. Transient local reactions, including erythema and induration beginning in the second week and lasting about one week occurred in more than half of the recipients. Systemic signs and symptoms, however, were not seen. Subsequent studies supported the safety and immunogenicity of the vaccine when given subcutaneously. Other important aspects of the vaccine include its inability to establish latency (140), to be isolated after immunization (including attempts from urine, saliva, and blood), and to induce antibody, lymphoproliferative (CD4þ) responses, and CD8þ cytolytic responses (141–144). Studies of Towne vaccine were also performed in renal transplant recipients because of the known risk associated with transplant, especially when an HCMV seronegative recipient receives a kidney from a seropositive donor. Three placebocontrolled, randomized studies revealed that Towne vaccine did not prevent HCMV infection but modified the severity of disease with severe disease reduced by *85% (145–148) (Table 3). A unique challenge study conducted in 1989 (149) provided further evidence for the protection offered by Towne vaccine (Table 4). In this study, healthy HCMV seronegative Catholic priests were immunized with vaccine or not immunized and then challenged with a range of doses of the Toledo strain of HCMV, a low passage isolate that retained virulence (150). Vaccinated subjects were protected from infection compared to unimmunized controls, but were not protected as well as those who were naturally immune prior to challenge with Toledo.

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Table 3 Comparative Results of Three Blinded Trials of Towne Vaccine in Seronegative Renal Transplant Patients Who Received Kidneys from Seropositive Donors Rate of all CMV disease (%)

Rate of severe CMV disease (%)

Trial

n

V

P

V

P

Pennsylvania Minnesota Multicentric All

67 35 61 163

39 33 38 37

55 43 59 54

6 5(10)a 0 3

35 36 17 29

Reduction of severe disease in vaccinated compared with placebo 84 87 100 89

a A 10% rate was reported in the original publication, but this includes one case that occurred subsequent to pancreatic transplant after a renal transplant free of CMV disease. Without that case, the incidence was 5%. Abbreviations: CMV, cytomegalovirus; P, patients given placebo; V, patients given vaccine. Source: From Ref. 147.

In a study of women naturally exposed to HCMV from their infant shedding HCMV in day care, Towne vaccine did not provide protection from infection, whereas those women with prior natural infection were protected from reinfection (151). However, in this study, one dose of Towne vaccine induced neutralizing antibody titers that were 10- to 20-fold less than after wild type infection. In a subsequent trial, using a different lot of Towne vaccine, neutralizing titers were comparable to natural infection (152). As the latest trial did not measure protection, it is unclear if a more potent Towne or other attenuated HCMV vaccine would provide better protection. Studies with larger doses of Towne vaccine continue. In an attempt to improve the immunogenicity of Towne vaccine, it has also been combined with interleukin 12 (IL-12) (143), which appeared to have some beneficial effects. Further, evidence for the utility of attenuated live virus vaccines comes from studies using a temperature sensitive, replication defective MCMV (153) or an attenuated MCMV, RV7 that contains a deletion of 7.7 kb spanning portions of MCMV HindIII-J and -I (154). RV7 replication was similar to wild type MCMV in vitro, but RV7 failed to replicate in target organs of immunocompetent BALB/c mice or severe combined immunodeficient mice (154,155). Following immunization with RV7, protection against a virulent salivary gland passaged virus was seen following parenteral as well as mucosal routes of challenge (155). Another approach to attenuate CMV has been to remove immune–evasive genes from CMV. In one proof of principle Table 4 Challenge with Low-Passage Toledo Strain Cytomegalovirus in Seronegative, Seropositive, and Towne-Vaccinated Participants (Number positive/Number inoculated) Dose Seronegative Illness Laboratory abnormalitiesa Infection Naturally seropositive Illness Laboratory abnormalities Infection Vaccinated Illness Laboratory abnormalities Infection

103

102

10 pfu

ND ND ND

2/2 2/2 2/2

4/4 4/4 4/4

3/5 5/5 3/5

0/5 0/5 1/5

0/2 0/2 1/2

ND ND ND

1/7 3/7 4/7

0/5 1/5 0/5

a Laboratory abnormalities include evidence of lymphocytosis, thrombocytopenia, or hepatitis.

evaluation, 32 genes were selectively removed from MCMV to produce a mutant that grew well in vitro but was 10,000 fold attenuated in highly susceptible, immunosuppressed mice (156). Recombinant Towne/Toledo An ideal live HCMV vaccine might combine the safety profile of Towne vaccine and the immunogenicity of wild type HCMV. If the Towne strain is overattenuated because of extensive mutations acquired during the 125 passages, then perhaps replacing defined regions of the Towne genome with the corresponding region from a wild type virus could modify this overattenuation. Indeed, researchers at MedImmune chose this approach and used the Toledo strain, a low passage isolate that had been previously characterized and tested in human volunteers as the wild-type challenge in studies of Towne vaccine (149,150) as the wild-type virus donor for recombination. They initially characterized genetic differences between Towne and Toledo and identified deletions in the Towne vaccine strain including the UL/b0 sequence, which is at the right edge of the unique long segment of the HCMV genome of Toledo and wild type isolates (157). They then constructed a set of four vaccine candidates (Fig. 3). Vaccine candidates were constructed by cotransfection of overlapping cosmid clones so that every region of the Towne genome was replaced in at least one candidate vaccine (158). For safety reasons, the genome of each cosmid is primarily derived from the genome of the Towne strain (&70%) and each still contains at least one of the defined Towne deletions. To increase immunogenicity, each construct also contains a Toledo replacement for at least one of the defined Towne deletions as well as the UL/b0 region of Toledo. Gene products of UL/b0 are responsible for tropism to macrophages and endothelial cells, and appear to improve immunogenicity. Because the UL/b0 of Toledo is in reverse orientation to wild-type HCMV, this can also serve as a marker allowing differentiation between vaccine and wild type HCMV. An initial placebo-controlled double-blind phase I clinical trial of these four vaccine candidates conducted in HCMV seropositive adults has been completed (159). The vaccines appeared to be safe, although some local reactogenicity was observed. Vaccine virus was not recovered from the immunized subjects. Only subsequent clinical trials in seronegative subjects can determine whether the right balance between safety and immunogenicity has been achieved in any of the four candidates. Although MedImmune is not currently pursuing this approach, a trial in CMV seronegative subjects is imminent (S. Adler, personal communications, 2007).

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Figure 3 Schematic representation of CMV chimeric strains generated by recombination of the TowneAV variant attenuated virus (gray) and the low passage virulent Toledo virus (black). The unique long (UL) and short (US) components are bracketed by inverted repeats b0 c0 and a0 c0 -ca. Boxes denote repeat sequences on both genomes. The unique sequence present in the Toledo genome are depicted as hatched segments and their orientation is denoted by the arrow in the Toledo genome. Abbreviation: CMV, cytomegalovirus.

Prime Boost The rationale for evaluating prime boost strategies with canarypox was largely developed by HIV researchers in the hope of achieving antibody and cell-mediated immune responses that were higher than with either vaccine alone (160). In the initial report evaluating HCMV prime boost strategies, Adler et al. (124) showed that priming with two doses of canarypox expressing HCMV gB induced only a weak response but primed for a subsequent boost by live attenuated Towne CMV vaccine. Subjects primed with the canarypox HCMVgB developed higher neutralization and gB ELISA titers that developed sooner and lasted longer than in Towne recipients who had first received a canarypox expressing a rabies protein. In a subsequent trial (161), the effect of priming with two doses of canarypox HCMVgB followed by two doses of the subunit HCMVgB vaccine with the adjuvant MF59 or the combination of canarypox HCMVgB plus simultaneous HCMVgB/MF59 did not have an advantage over immunization with three doses of gB/MF59. Although serum neutralizing antibody responses developed more quickly after priming with the canary pox vector, neutralization and ELISA gB titers, lymphoproliferative and g interferon response were all equivalent after the final dose of vaccine. Thus, it appears that the potent combination of gB and MF59 induces substantial levels of antibody and cell mediated immune responses to HCMV gB that were not enhanced by previous or simultaneous canarypox HCMVgB immunizations.

DNA and Other Vaccine Approaches DNA vaccines (genetic immunization) have become a popular concept, but initial optimism stemming from small animal evaluations has been tempered by the poorer immunogenicity seen in larger mammals including humans. Several studies have reported both cell-mediated and humoral responses to DNA immunization with either CMV gB or pp65 plasmids in mice and guinea pigs (162–165). In one study, immunization with a plasmid expressing the MCMV immediate early gene (IE1), the major CD8 CTL target provided some but incomplete

protection against a lethal MCMV infection, and more consistent protection against a sublethal MCMV challenge (165). In another report, the same group evaluated plasmids encoding MCMV homologs of HCMV, including the tegument (M32, M48, M56, M82, M83, M69, and M99), capsid (M85 and M86) and nonstructural antigens IE1-pp89 and M84. Only pp89 and M84, a nonstructured protein that shows homology with HCMV UL83-pp65 provided protection (166). This group went on to show that DNA immunization with gp34 also provided protection in a murine model and that immunization with a pool of 10 MCMV genes that were not individually protective did provide protection against low-tomoderate virus challenges (167). Combining the 10 genes with the three previously shown to be individually protective (gp34, pp89 and M84) improved protection. The highest level of protection was, however, seen when immunization with this pool of 13 genes was followed by immunization with formalininactivated MCMV. This immunization regimen reduced viral titers following both systemic (167) and mucosal (intranasal) challenge (168). Most recently, a DNA vaccine expressing the gcII antigens gM and gN was shown to be immunogenic in mice and rabbits (101). When combined, the gN and gM vaccine induced antibody to both gM and the gcII complex and higher neutralizing antibody titers than the gM or gN vaccine alone. Neutralizing antibody to the homologous AD169 strain as well as to the heterologous strains Towne and Davis were induced. Given the abundance of the gM protein (46) and the heterogeneity of gB as discussed above, the gM/gN complex may provide additional protection when added to other vaccine candidates such as gB. The evaluation of gB and pp65 expressing DNA vaccines has recently been extended to the rhesus macaque model (169). Animals were immunized with either a secreted form of rhesus monkey CMV gB and pp65 or this combination with a viral interleukin-10 DNA vaccine. Animals developed antibody to gB and pp65 and a weaker response to IL-10. Immunization decreased peak viral titers in the blood in both immunized groups.

Chapter 59: Cytomegalovirus Vaccines

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Table 5 Status of Cytomegalovirus Vaccine Candidates Experimental Vaccine

Comments

Status

Vaccines in Recent Clinical Trials Live attenuated Towne strain Safe and well tolerated Does not produce viremia or latency Induces cell mediated immunity and antibody Towne/Toledo chimera Safe in seropositive subjects No viremia in seropositive subjects Glycoprotein B þ MF59 Provided protection from infection in young women Local side effects Induced neutralizing antibody VEE replicon gB, pp65, IE1 Safe Induced neutralizing antibody and T cell responses DNA vaccine Safe Moderately immunogenic Vaccines in preclinical trials Vectored vaccines Peptide vaccines DNA vaccines Dense body Formalin inactivated virus

Vaccinia and adenovirus: gB, pp65, IE1 pp65, IE1 gB, pp65, IE1 All structural proteins All structural proteins

A bivalent CMV DNA vaccine, VCL-CB01, consisting of 2 plasmids, VCL-6368 and VCL-6365 (Vical Inc. San Diego, California, U.S.) formulated with poloxamer CRL1005 and BAK in PBS has recently been evaluated in a phase 1 clinical trial. Doses of 1 mg and 5 mg were evaluated in 22 CMV-seropositive and 22 CMV-seronegative adults (170). VCL-6368 encodes pp65 from AD169 with the putative protein kinase domain removed by deletion of aa 435–438. VCL-6365 encodes the extracellular domain (aa 1–713) of CMV gB. (169). Overall, the vaccine was well tolerated, with no serious adverse events. Immunogenicity, as measured by enzyme-linked immunosorbant assay and/or ex vivo interferon (IFN)–g enzyme-linked immunospot assay, was documented in 45.5% of CMV-seronegative subjects and in 25.0% of CMV-seropositive subjects while 68.1% of CMV-seronegative subjects had memory IFN-g T cell responses induced. In CMVseropositive subjects, VCL-CB01 boosted existing pp65 T cell responses but not gB antibody responses. Thus, this CMV vaccine appears to be more effective for inducing CMV antigen–specific T cells than gB-specific antibody. Another intriguing approach for CMV immunization is the use of dense bodies, defective noninfectious enveloped particles that are produced spontaneously in cell culture, and which contain all the viral structural proteins. Pepperl et al reported that immunization of animals with dense bodies induced both humoral and CTL responses (171,172). Clinical trials in Europe are anticipated.

CONCLUSIONS Despite the obvious need for a CMV vaccine, development has lagged compared to other pathogens with an equal or lesser burden of disease. However, there have been continued efforts to develop vaccines with traditional and more modern vaccine approaches (Table 5). The recent initiation of phase I and II clinical trials by an enlarging number of developers is encouraging, and heralds a renewed interest and dedication to the production of an effective CMV vaccine.

Evaluated in renal transplants and healthy adults Protection following challenge Not protective in day care setting Only evaluated in seropositive subjects Study in seronegative subjects being discussed Phase II trial completed in young women Two additional efficacy trails in progress (one in young females, one in transplant) Initial phase I trial completed in CMV seronegative adults Initial phase I trial complete

Induce cell mediated and antibody mediated Induce cell mediated and antibody mediated Induce cell mediated and antibody mediated Induce cell mediated and antibody mediated Boost response to DNA vaccine

immunity immunity immunity immunity

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114. Nicholson KG, Colegate AE, Podda A, et al. Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/ Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza. Lancet 2001; 357: 1937–1943. 115. Pass RF, Zhang C, Evans A, et al. Vaccine prevention of maternal cytomegalovirus infection. N Engl J Med 2009; 360(12):1191–1199. 116. Stanberry LR, Rosenthal SL, Mills L, et al. Longitudinal risk of herpes simplex virus (HSV) type 1, HSV type 2, and cytomegalovirus infections among young adolescent girls. Clin Infect Dis 2004; 39:1433–1438. 117. Tackaberry ES, Dudani AK, Prior F, et al. Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco. Vaccine 1999; 17:3020–3029. 118. Paston SJ, Dodi IA, Madrigal JA. Progress made towards the development of a CMV peptide vaccine. Hum Immunol 2004; 65:544–549. 119. Diamond DJ, York J, Sun JY, et al. Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997; 90:1751–1767. 120. La Rosa C, Krishnan R, Markel S, et al. Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries. Blood 2001; 97:1776–1786. 121. Longmate J, York J, La Rosa C, et al. Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes. Immunogenetics 2001; 52:165–173. 122. Plotkin SA, Cadoz M, Meignier B, et al. The safety and use of canarypox vectored vaccines. Dev Biol Stand 1995; 84:165–170. 123. Gonczol E, Berensci K, Pincus S, et al. Preclinical evaluation of an ALVAC (canarypox)–human cytomegalovirus glycoprotein B vaccine candidate. Vaccine 1995; 13:1080–1085. 124. Adler SP, Plotkin SA, Gonczol E, et al. A canarypox vector expressing cytomegalovirus (CMV) glycoprotein B primes for antibody responses to a live attenuated CMV vaccine (Towne). J Infect Dis 1999; 180:843–846. 125. Berencsi K, Gyulai Z, Gonczol E, et al. A canarypox vectorexpressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMVseronegative subjects. J Infect Dis 2001; 183:1171–1179. 126. Berencsi K, Rando RF, deTaisne C, et al. Murine cytotoxic T cell response specific for human cytomegalovirus glycoprotein B (gB) induced by adenovirus and vaccinia virus recombinants expressing gB. J Gen Virol 1993; 74(pt 11):2507–2512. 127. Marshall GS, Ricciardi RP, Rando RF, et al. An adenovirus recombinant that expresses the human cytomegalovirus major envelope glycoprotein and induces neutralizing antibodies. J Infect Dis 1990; 162:1177–1181. 128. Jonjic S, del Val M, Keil GM, et al. A nonstructural viral protein expressed by a recombinant vaccinia virus protects against lethal cytomegalovirus infection. J Virol 1988; 62:1653–1658. 129. Wang Z, La Rosa C, Maas R, et al. Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus. J Virol 2004; 78: 3965–3976. 130. Shanley JD, Wu CA. Intranasal immunization with a replicationdeficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity. Vaccine 2005; 23:996–1003. 131. Rist M, Cooper L, Elkington R, et al. Ex vivo expansion of human cytomegalovirus-specific cytotoxic T cells by recombinant polyepitope: implications for HCMV immunotherapy. Eur J Immunol 2005; 35:996–1007. 132. Del Val M, Schlicht HJ, Volkmer H, et al. Protection against lethal cytomegalovirus infection by a recombinant vaccine containing a single nonameric T-cell epitope. J Virol 1991; 65: 3641–3646.

Chapter 59: Cytomegalovirus Vaccines 133. Wang Z, La Rosa C, Lacey SF, et al. Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection. J Clin Virol 2006; 35:324–331. 134. Rayner JO, Dryga SA, Kamrud KI. Alphavirus vectors and vaccination. Rev Med Virol 2002; 12:279–296. 135. Pushko P, Parker M, Ludwig GV, et al. Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 1997; 239: 389–401. 136. Reap EA, Dryga SA, Morris J, et al. Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1 and gB proteins. Clin Vaccine Immunol 2007; 14(6):748–755. 137. Bernstein DI, Reap EA, Katen K, et al. Randomized, double-blind, phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers. Vaccine (in press). 138. Marshall GS, Plotkin SA. Progress toward developing a cytomegalovirus vaccine. Infect Dis Clin North Am 1990; 4:283–298. 139. Plotkin SA, Farquhar J, Horberger E. Clinical trials of immunization with the Towne 125 strain of human cytomegalovirus. J Infect Dis 1976; 134:470–475. 140. Plotkin SA, Huang ES. Cytomegalovirus vaccine virus (Towne strain) does not induce latency. J Infect Dis 1985; 152:395–397. 141. Starr SE, Glazer JP, Friedman HM, et al. Specific cellular and humoral immunity after immunization with live Towne strain cytomegalovirus vaccine. J Infect Dis 1981; 143:585–589. 142. Gonczol E, Ianacone J, Furlini G, et al. Humoral immune response to cytomegalovirus Towne vaccine strain and to Toledo low-passage strain. J Infect Dis 1989; 159:851–859. 143. Jacobson MA, Sinclair E, Bredt B, et al. Safety and immunogenicity of Towne cytomegalovirus vaccine with or without adjuvant recombinant interleukin-12. Vaccine 2006; 24:5311–5319. 144. Jacobson MA, Sinclair E, Bredt B, et al. Antigen-specific T cell responses induced by Towne cytomegalovirus (CMV) vaccine in CMV-seronegative vaccine recipients. J Clin Virol 2006; 35: 332–337. 145. Plotkin SA, Smiley ML, Friedman HM, et al. Towne-vaccineinduced prevention of cytomegalovirus disease after renal transplants. Lancet 1984; 1:528–530. 146. Balfour HH Jr. Prevention of cytomegalovirus disease in renal allograft recipients. Scand J Infect Dis Suppl 1991; 80:88–93. 147. Plotkin SA, Higgins R, Kurtz JB, et al. Multicenter trial of Towne strain attenuated virus vaccine in seronegative renal transplant recipients. Transplantation 1994; 58:1176–1178. 148. Plotkin SA. Vaccination against cytomegalovirus, the changeling demon. Pediatr Infect Dis J 1999; 18:313–325; quiz 326. 149. Plotkin SA, Starr SE, Friedman HM, et al. Protective effects of Towne cytomegalovirus vaccine against low-passage cytomegalovirus administered as a challenge. J Infect Dis 1989; 159:860–865. 150. Quinnan GV Jr., Delery M, Rook AH, et al. Comparative virulence and immunogenicity of the Towne strain and a nonattenuated strain of cytomegalovirus. Ann Intern Med 1984; 101:478–483. 151. Adler SP, Starr SE, Plotkin SA, et al. Immunity induced by primary human cytomegalovirus infection protects against secondary infection among women of childbearing age. J Infect Dis 1995; 171:26–32. 152. Adler SP, Hempfling SH, Starr SE, et al. Safety and immunogenicity of the Towne strain cytomegalovirus vaccine. Pediatr Infect Dis J 1998; 17:200–206. 153. Gill TA, Morley PJ, Sweet C. Replication-defective mutants of mouse cytomegalovirus protect against wild-type virus challenge. J Med Virol 2000; 62:127–139. 154. Cavanaugh VJ, Stenberg RM, Staley TL, et al. Murine cytomegalovirus with a deletion of genes spanning HindIII-J and -I displays altered cell and tissue tropism. J Virol 1996; 70:1365–1374. 155. MacDonald MR, Li XY, Stenberg RM, et al. Mucosal and parenteral vaccination against acute and latent murine cytomegalovirus

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60 Epstein–Barr Virus Vaccines Viviana P. Lutzky and Denis J. Moss The EBV Biology Laboratory, Division of Infectious Diseases and Immunology, The Queensland Institute of Medical Research, Queensland, Australia

INTRODUCTION Epstein–Barr virus (EBV) is a common herpes virus and a significant cause of human disease. During primary infection, colonization of the lymphoid system occurs through virusdriven expansion of infected B cells, which selectively express six latent EBV proteins (EBNAs 1, 2, 3, 4, 6, –LP). Viral shedding into the oropharynx is another feature of primary EBV infection and arises from expression of the ‘‘lytic switch’’ protein BZLF1, which launches the lytic cycle cascade including gp85, BMLF1, BMRF1, BHRF1, and gp350 proteins (1). EBV infection reaches an equilibrium with its human host so that the majority of individuals infected with the virus remain lifelong secretors in the absence of any clinical symptoms. However, in certain circumstances this benign virus is a significant cause of disease, although it is likely that in these instances pathogenesis is a ‘‘mistake’’ of nature or arises as a result of clinical intervention such as the use of immunosuppressive drugs. It has been convenient to classify EBVassociated diseases in terms of the degree of expression of the EBV-latent proteins. Thus these diseases are frequently referred to as latency III, latency II, or latency I depending on all (latency III), some (latency II), or one (latency I) latency proteins are expressed (Fig. 1). From both a scientific and commercial perspective, there is relatively strong justification for the development of a prophylactic vaccine to prevent the clinical symptoms of primary EBV infection (infectious mononucleosis, IM), which affects about 10% of individuals in some developed countries. The justification for this vaccine is enhanced by the fact that this same vaccine may protect EBV seronegative transplant recipients who are at risk of developing posttransplant lymphoproliferative disease (PTLD) and EBV seronegative young males at high risk of X-linked lymphoproliferative (XLP) disease. In seronegative heart and lung transplant recipients, PTLD occurs with an incidence of about 10%, while the incidence of XLP is rare, although associated with a high mortality. For reasons outlined below, a separate vaccine formulation would be required for nasopharyngeal carcinoma (NPC), which is relatively common in southern China and in many parts of Southeast Asia. Indeed, in many of these areas, NPC is the commonest form of cancer in males of 30 to 45 years. The fact that the same vaccine formulation may also find application in Hodgkin’s lymphoma (HL), which is often strongly associated with EBV, provides justification for a combined NPC/HL vaccine, although the scientific basis for such a

vaccine is less compelling than in the case of IM/PTLD/XLP (see the following sections).

BIOLOGY AND IMMUNOLOGY OF EBV RELEVANT TO VACCINE FORMULATIONS As with other gamma herpesviruses, EBV encodes a large set of lytic cycle genes together with a number of latent genes that are associated with expansion of the latent EBV pool in B lymphocytes (1). The virus gains entry into the body by infection of B lymphocytes in the oral cavity via an interaction between the major viral glycoprotein gp350 and the complement receptor CR2, which is expressed on B cells. It seems likely that one of earliest detectable events following primary infection is the expression of lytic cycle proteins resulting in the release of infectious virus into the oral cavity followed by a generalized seeding of EBV-infected B lymphocytes, which express a family of latent proteins, EBV nuclear protein (EBNA) 1, 2, 3A, 3B, 3C, and latent membrane proteins (LMP) 1, 2A, and 2B (1). Primary infection with EBV results in symptoms of acute IM in about 50% of adolescents and is coincident with a marked lymphocytosis (dominated by EBV-specific cytotoxic CD8þ T cells) and the appearance of an immunoglobulin M (IgM) response to a variety of EBV proteins, most notably the capsid antigen of the virus. Current evidence suggests that this cytotoxic T-cell (CTL) response, which includes both CD4þ and CD8þ T cells, restricts expansion of these latently infected B cells and results in a long-term carrier state in which there is an equilibrium between the level of secretion of the virus and the number of latently infected B cells. In instances where this equilibrium is disturbed as a result of immunosuppression, for example, in transplant patients, the number of virus-infected B cells increases the risk of PTLD particularly in EBV seronegative graft recipients. The malignant cells in NPC and HL express a more restricted set of EBV genes (Fig. 1A) including LMP1, LMP2, EBNA1, EBER transcripts, and the BamHI A RNAs (1), although the degree of expression of the LMP proteins appears variable within tissue and between biopsies. It should be pointed out that the extreme sensitivity of T cells to recognize peptide epitopes bound to major histocompatibility complex (MHC) compared with the sensitivity of antibody to recognize protein using conventional assays means that this apparent variable level of LMP proteins detected in NPC may not be relevant when considering vaccine formulations.

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Figure 1 Influence of EBV gene expression in EBV-associated diseases on potential strength of CTL response to epitopes within EBV lytic and latent proteins. (A) EBV gene expression in each of the EBV-associated diseases. The latency III disorders, including infectious mononucleosis, posttransplant lymphoproliferative disease, and X-linked lymphoproliferative express EBNAs 1, 2, 3A, 3B, 3C, and LMP 1 and 2. The latency II tumors nasopharyngeal carcinoma and Hodgkin’s lymphoma express EBNA1, LMP1, and LMP2. BL is a latency I tumor that only expresses EBNA 1. Furthermore, latency I and II malignancies often have downregulated expression of major histocompatibility complex class II, TAP 1 and 2, and ICAM 1 molecules, thus reducing both the processing and cell surface expression of EBV proteins. (B) The relative strength of CTL responses against different EBV proteins indicates that the dominant CTL responses are directed against the lytic and latent proteins EBNA 3A, 3B, and 3C, while the response to epitopes within LMP1 and 2 are significantly reduced. Abbreviations: EBV, Epstein-Barr virus; CTL, cytotoxic T cell; EBNA, EBV nuclear protein; LMP, latent membrane proteins; BL, Burkitt lymphoma; TAP, transporter associated with antigen processing; ICAM, intercellular adhesion molecule.

The marked expansion of CD8 T cells during primary infection and the ease of detection and analysis of these responses following remission of symptoms have focused interest on these responses at the expense of CD4þ responses. It would be a mistake to infer that CD4 responses are not important and that antigens to elicit such responses should not be considered for inclusion in vaccine formulations. Nevertheless, it is not clear whether the primary function of the CD4 cells is in maintenance of CD8 T cell immunity or whether they are capable of exerting an additional effector role. There is certainly an impression among investigators that CD4 cells have a helper role in the in vitro expansion and preservation of EBV CTL used for adoptive transfer into PTLD patients. Other recent studies have described the isolation of LMPspecific CD4þ T-cell clones that can efficiently recognize infected targets and prevent their outgrowth (2). Although there has been a suggestion in the literature that CD4 cells with apparent EBNA1 specificity are able to recognize and kill latently infected target cells, it is unclear how EBNA1 is being processed and presented by the human leukocyte antigen (HLA) class II pathway to CD4 T cells (3–5). Recent work has shown that some EBNA1 epitopes are presented on EBVinfected cells at sufficient level for CTL recognition (6).

OPPORTUNITIES AND CHALLENGES INHERENT IN EBV VACCINE FORMULATIONS The phenotype of the virus-infected cell in IM and PTLD appears favorable in terms of the prospects of developing an effective vaccine. Thus, the virus-infected cells in these cases have upregulated expression of MHC class I and II, a favorable surface phenotype (ICAM 1 and 2 and LFA3), processing proteins (TAP 1 and 2), and express the immunodominant proteins EBNA 3A, 3B, and 3C (Fig. 1B). However, one of the confounding problems with regard to a vaccine to NPC and HL is that both of these malignancies are characterized by the lack

of expression of these immunodominant EBV proteins. It appears that EBV gene expression is confined to EBNA1, LMP1, and LMP2 in the case of HL, while in the case of NPC, there is variable expression of both LMP proteins (Fig. 1A). A further difficulty in relation to a vaccine to NPC, and to some extent to HL, is the fact that the environment of the malignancy includes negative elements that render the establishment and maintenance of a meaningful immune response somewhat difficult. Thus, for instance, it has been established that HL tumors express transforming growth factor beta (TGFb) and interleukin-10 (IL-10), together with the thymus-and activation-regulated chemokine (TARC) (which is associated with a T-helper 2 [TH2] phenotype), although this does not appear to be the case for NPC (7). Despite these reservations, it appears that the best hope of developing an NPC or HL vaccine lies in boosting the CTL response to LMP1 and LMP2 to a level that can overwhelm these negative regulators of a protective CTL response.

EXPERIMENTAL VACCINES It is unfortunate that there is no model of EBV infection in animals which entirely reproduces the cell-virus relationships seen in symptomatic primary infection in humans and upon which vaccine-formulation decisions could be based. The Rag2/ gc/mutant strain of mice that lacks B, T, and NK cells, were engineered to develop a functional human immune system following intrahepatic transfer of CD34þ human cord blood cells into newborns (8). EBV successfully infected the human immune cells in these Hu-Rag2-gc and these mice developed T cells that proliferated in response to EBV antigen in vitro. While it remains to be clarified how human T-cell selection on a mouse thymic background occurs, the T cells generated discriminate self from allogeneic MHC and these xenotransplanted mice raised human IgG to tetanus toxoid antigen. Hu-Rag2-gc mice thus offer an opportunity to test EBV vaccine formulations in a human

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cell background that supports efficient high-level viral gene expression. With the Hu-Rag2-gc mice xenotransplant model, it may be feasible to evaluate (i) the immune response to EBV in the context of the human immune response over a relatively short period of time and (ii) the safety and efficacy of novel vaccine formulations in humans. Human HLA transgenic mice have been used to test the ability of various EBV vaccine formulations to induce CTL responses. Particular use has been made of HLA A2 transgenic mice, which express a chimeric class I molecule composed of the a-1 and -2 domains of the human A*0201 allele and the a-3 domains of the mouse H-2Kb class I molecules (9). These mice can be used to assess the ability of potential formulations to activate EBV-specific CTL responses and secondly these activated T cells are subsequently available to determine the efficacy of these CTL to resist the expansion of EBV-driven malignancies expanding in severe combined immunodeficiency (SCID) mice. Nevertheless, it should not be assumed that the relative dominance between individual CTL epitopes seen in response to immunization of human HLA transgenic mice will reflect immunodominance seen in response to vaccination of humans. The best primate model is based on infection of rhesus monkeys with the rhesus lymphocryptovirus (LCV). This virus, which shares significant sequence homology with EBV, reproduces many of the key events associated with primary EBV infection when these primates are infected orally. Moreover, this model appears to be relevant to the testing of vaccine formulations since animals show resistance to a second challenge following primary infection (10). Although, this model is likely to prove expensive when used to screen potential formulations, it may provide an excellent system for demonstrating final confirmation of the efficacy of a formulation before human trials begin.

Vaccines Associated with Primary EBV Infection About 10% of individuals living in Western countries contract the symptoms associated with IM. There are two schools of

thought regarding the strategy that should be used with regard to a prophylactic vaccine. The first revolves around formulations incorporating the lytic protein gp350, which are known to protect cotton-top tamarins from developing lymphomas following intraperitoneal inoculation with high titered EBV (Table 1). Such a vaccine has been used in China when formulated in vaccinia and indeed a proportion of the EBV seronegative individuals appeared to be protected from primary infection (11). In contrast, a more recent gp350 formulation suggested that the symptomatology of primary infection might be reduced but that the rate of seroconversion was unaltered (12). An alternative strategy is to use formulations incorporating EBV latent proteins or immunogenic determinants within these proteins. This approach is supported by the observations of a patient treated by adoptive immunotherapy with autologous CTL, in which it was noted that there was a correlation between the induction of a strong latent antigen-specific response and the cessation of disease, while a sustained lytic response was coincident with disease progression (31). Secondly, a study of HLA identical individuals, one of whom sustained prolonged clinical symptoms from primary EBV infection and the other who recovered after a brief period, has been useful in ascribing the link between the CTL response and the severity of acute symptomatology. It was clear that rapid recovery was associated with the induction of a broad latent antigen-specific response and that acute disease corresponded with a sustained and focused lytic-antigen response (32). Thus, provided that we can assume that the lessons from EBV infection of B lymphocytes in vitro are relevant in vivo and that the initial event in primary infection is contact between the virus and a B lymphocyte, a strong case can be mounted in favor of directing an IM vaccine toward latent rather than lytic proteins. Such a vaccine would restrict the latently infected B cell pool expansions, some of which presumably progress toward expression of lytic proteins, which appear to be responsible for the lymphocytosis associated with acute infection.

Table 1 Vaccine Formulations and Immunotherapy Attempts Vaccine or immunotherapy target Latency III Primary EBV infection prophylactic Primary EBV infection Prophylactic Primary EBV infection prophylactic Primary EBV infection Seronegative graft recipient prophylactic vaccine X-linked lymphoproliferative PTLD patient Prophylactic transplant recipient PTLD patient High EBV DNA prophylactic PTLD patient Latency II Advanced NPC Local recurrence NPC NPC, therapeutic Advanced HL Relapsed HL

Vaccine/immunotherapy formulation options

References

gp350 vaccinia recombinant gp350 formulated adjuvant peptide vaccine in adjuvant Latent proteins formulated as a polytope in an adjuvant T-cell peptide epitope/tetanus toxoid/Montanide ISA 720 Formulations above potentially applicable Formulations above potentially applicable Donor EBV CTL T depleted Autologous EBV CTL Autologous EBV CTL Autologous EBV CTL Allo BMT T cell depleted

11 12–15

CTL by auto LCL activation Latent membrane proteins 2 peptide-pulsed DCs LCL-stimulated allo CTL LCL stimulated auto CTL Auto LCL Haploidentical EBV CTL LCL-stimulated CTL

23,24 25 26 27 28 29 30

13,16 17 As above As above 18 19–21 18,22 20 18

Abbreviations: EBV, Epstein-Barr virus; PTLD, posttransplant lymphoproliferative disease; CTL, cytotoxic T cell; NPC, nasopharyngeal carcinoma; HL, Hodgkin’s lymphoma; BMT, bone marrow transplantation; LCL, lymphoblastoid cell line; DC, dendritic cell.

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Possible formulations based on latent proteins have been reviewed previously (13,33) and include the use of latent protein peptide epitopes either alone or as a polyepitope formulated in a suitable adjuvant. It seems that the threat of litigation will preclude the biotechnology industry developing a preventative IM vaccine, which involves the use of a live delivery vector in view of the group at highest risk (seronegative middle-class adolescents in developed countries). A successful vaccine for primary EBV infection would also find application in the prevention of PTLD since it is well established that EBV seronegative graft recipients are at a significantly increased risk of developing disease compared with EBV seropositive individuals. Likewise, young EBV seronegative males at risk of XLP should be protected from developing latency III tumors to which they are particularly prone.

Vaccines for NPC and HL The strategy for latency II malignancies needs to take into account the fact that only the weakly immunogenic LMP proteins are expressed. A number of new technologies are in preclinical testing and development phase. Representative examples of technologies and references appear in Table 1. It is entirely possible that a live vaccine will be used both in the case of those identified at maximal risk and those with existing disease. Thus, a polyepitope incorporating multiple LMP CTL epitopes and delivered in a replication-deficient vector is able to cure LMP-expressing tumors in mice (9,34). Another technology currently under development is a scrambled antigen vaccine (SAVINE) (35). This technology is designed to incorporate LMP and EBNA1 sequences as overlapping peptides, rearranged and rejoined in such a way that potential CD4þ and CD8þ CTL epitopes are retained for safer vaccine delivery. When formulated in a live vector, this technology may overcome the problems inherent in the utility of a polyepitope, since all of the immunogenic determinants will be included irrespective of HLA type of the vaccine recipient. Codelivery of immune-potentiating cytokines and specific targeting of recombinant antigens through the MHC class I/II pathways is likely to enhance the efficacy of these vaccines (16).

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THE ROLE OF ADOPTIVE IMMUNOTHERAPY Adoptive immunotherapy of either autologous or allogeneic EBV-specific CTL has and will have an important role in establishing scientific principles and might find clinical application in conjunction with conventional therapies. Adoptive immunotherapy in this context involves the ex vivo activation of EBV-specific T cells, thus circumventing in vivo host immune-suppressive mechanisms and makes available large numbers of virus-specific CTL for administration into either an autologous or allogeneic host (Fig. 2). Although this therapy has been used in a number of centers for either prevention or treatment of PTLD (18–20,36), recent advances suggest that it may find either prophylactic or therapeutic application in NPC and HL (25). A recent report on a trial of immunotherapy with autologous EBV-targeted CTLs in NPC stage IV patients showed an enhancement of EBV-specific immune responses together with clinical responses (23). A major constraint to the wider application of autologous CTL-based therapy is the delay between diagnosis and the preparation of a therapeutic dose of T cells. To overcome this limitation, banks of CTLs derived by ex vivo activation of effector cells from either haploidentical or HLA-shared individuals from healthy seropositive subjects can be established (22). This strategy has proven clinical efficacy and offers a distinct logistic advantage of speed of access and ease of generation that might permit its wide-scale use in treating both PTLD and other malignancies. This may be particularly relevant for developing countries where T cells could be transported to remote locations in which access to standard chemo/radiotherapy is restricted. Most importantly, infusion of haploidentical allogeneic CTL is associated with low rates of graft-versus-host disease, suggesting that prolonged in vitro culture diminishes alloreactivity. An innovative approach to overcome the time between diagnosis and the availability of CTL has recently been demonstrated using retroviral vectors encoding the relevant T-cell receptor to infect PBMCs. Potentially, this might dramatically decrease the time taken to prepare such T-cell preparations, although the use of retroviral vectors in humans is likely to present a formidable barrier to the wide-spread use of this technique (37,38). Another significant challenge of adoptive immunotherapy is reversing the evasion strategies adopted by many

Figure 2 Adoptive immunotherapy in EBV-related diseases. This figure illustrates a T-cell-based adoptive immunotherapy for EBV-associated malignancies. Abbreviation: EBV, Epstein-Barr virus.

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tumors. This is particularly the case in relation to latency II malignancies where there is an environment that appears hostile to effective CTL control. Indeed, the Reed-Stermberg cells that comprise the malignant cells of HL use several strategies to create a protected immunological environment. They secrete TGF-b and IL-10 that can induce anergy of professional antigen-presenting cells and effector T cells (39); they can produce the chemokine TARC that attracts effector T cells with Th2-like phenotype (40) and they can express Fas-ligand that can induce apoptosis of activated T cells (41). More recently, it has been reported that ‘‘regulatory T cells’’ surrounding the tumor may play a significant protective role against the immune-mediated elimination of tumor cells (42,43). This barrier might be reversed by transduction of EBV CTL with a dominant TGF-b receptor (39) or by depletion of regulatory T cells (44). Alternatively, it might be possible to blockade the tumor microenvironment with cytokines such as IL-10, IL-13, and TGF-b (45). In another approach, CTL have also been retrovirally transduced to produce IL-12 to be more functional in a Th2-like environment and to induce a breakdown in this microenvironment that both protects and supports the tumor cells (46). Finally, CTL can also be induced to become more resistant to the Fas/FasLinduced apoptosis, by a retrovirally mediated production of small interfering RNA down modulating the Fas receptor (47). The future of adoptive immunotherapy as a treatment option is likely to be focused of malignancies occurring in Western countries since the cost of the infrastructure surrounding this technology is very significant. Thus, in practical terms, PTLD and HL are the most likely applications of this form of therapy.

INTEGRATION OF IMMUNOTHERAPY AND CONVENTIONAL CLINICAL CONTROL PROTOCOLS It is entirely probable that the various forms of immunotherapy will be integrated into conventional treatment protocols rather than be stand-alone solutions. Thus, in the case of transplant

recipients, a combination of vaccination, rituximab, EBV DNA screening, adoptive immunotherapy, reduction in the level of immunosuppression might all have a role in the prevention and treatment of PTLD (Fig. 3). Although rituximab is the preferred method of treatment for this malignancy, the emergence of tumor escape mutants remains a concern (48). The application of the therapies either alone or in combination (Fig. 3) may well offer the best opportunity of controlling PTLD. Despite the cost of adoptive immunotherapy, the best chance for the widespread use of this technology lies in the establishment of a worldwide CTL bank, which can be drawn on as required (49). In the case of NPC, prophylactic screening and posttherapy monitoring of individuals at high risk would appear to offer considerable advantages in areas when this malignancy is common (Fig. 4). There has been considerable debate on the relative advantages of the use of EBV DNA screening and IgA serology to predict patients at high risk of developing disease or detecting relapse. Plasma EBV DNA levels correlate with disease stage in NPC and return from pretreatment levels of thousands of copies per milliliter to low or undetectable levels after successful treatment. Although IgA serology is predictive of NPC, it cannot however be used to predict tumor staging or to monitor for disease relapse, as unlike EBV DNA levels the titer does not return to baseline after treatment (50). Several studies have demonstrated that there is no correlation between IgA viral capside antigen (VCA) serology and viral load in NPC patients, and that DNA studies are a more reliable indicator for predicting recurrent disease (51,52). Furthermore, EBV DNA levels in the plasma are an effective monitoring method for NPC recurrence (53). It is possible that the vaccination strategies outlined in Figure 4 could be used at different stages of NPC development and recurrence. Thus, the vaccine used by individuals without disease but with elevated levels of EBV DNA may be less aggressive than those with recurrent disease where the use of concurrent cytokine treatment or CD45 depletion may be justified to reverse the unfavorable tumor environment.

Figure 3 Integration of screening and vaccination into treatment and prevention of posttransplant lymphoproliferative disease. This figure illustrates a model for the management of EBV seronegative transplant recipients and includes a scheme for the integration of vaccination, conventional treatment with rituximab and monitoring EBV DNA levels at certain critical points. It also describes how adoptive immunotherapy might be initiated. Abbreviation: EBV, Epstein-Barr virus.

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Figure 4 The role of an NPC vaccine and associated treatment in the management of NPC. This figure illustrates a scheme for the integration of conventional NPC therapy with Epstein-Barr virus DNA screening of individuals at high risk together with the critical points for the use of prophylactic and therapeutic vaccination and adoptive immunotherapy. Abbreviation: NPC, nasopharyngeal carcinoma.

CONCLUSIONS Extensive studies on the immune response to EBV now give reason for serious consideration of an EBV vaccine. It is likely that the earliest therapeutic vaccines will be directed toward the EBV-associated malignancies; although, from a scientific point of view there is probably greater justification for testing formulations to prevent the symptoms of IM. Despite the sometimes impressive results reported from trials based on the adoptive transfer of either autologous or allogeneic EBV-specific CTL, this procedure is more likely to be used as a means of establishing scientific principles rather than having wide-spread clinical application. Therefore, an EBV vaccine(s) will not only be pivotal in treating existing EBV-related cancers, it will potentially prevent the onset and relapse of such diseases, for example, in countries where NPC is endemic. Such a vaccine will be a more economically viable option that is available to entire populations, unlike specialized treatments currently under development. Novel approaches are being developed to enhance the potency of EBV-specific CTL by targeting CTL to subdominant EBV proteins and by modifying CTL to render them resistant to the evasion strategies adopted by many tumors. The challenge remains to construct a vaccine that induces both CD8þ and CD4þ EBV T-cell immunity in individuals irrespective of their HLA type, using a vector that poses minimal risk.

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15. Gilbert K. Results of Phase I Clinical Trial for Epstein-Barr Virus Vaccine. Mountain View, CA: Aviron Press Release, 1999. 16. Thomson SA, Elliott SL, Sherritt MA, et al. Recombinant polyepitope vaccines for the delivery of multiple CD8 cytotoxic T cell epitopes. J Immunol 1996; 157:822–826. 17. Elliott SL, Suhrbier A, Miles JJ, et al. Phase I trial of a CD8þ T-cell peptide epitope-based vaccine for infectious mononucleosis. J Virol 2008; 82:1448–1457. 18. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92: 1549–1555. 19. Haque T, Amlot PL, Helling N, et al. Reconstitution of EBVspecific T cell immunity in solid organ transplant recipients. J Immunol 1998; 160:6204–6209. 20. Comoli P, Labirio M, Basso S, et al. Infusion of autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication. Blood 2002; 99:2592–2598. 21. Khanna R, Bell S, Sherritt M, et al. Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc Natl Acad Sci U S A 1999; 96:10391–10396. 22. Haque T, Wilkie GM, Taylor C, et al. Treatment of Epstein-Barrvirus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells. Lancet 2002; 360:436–442. 23. Comoli P, Pedrazzoli P, Maccario R, et al. Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein-Barr virustargeted cytotoxic T lymphocytes. J Clin Oncol 2005; 23(35):8942–8949. 24. Chua D, Huang J, Zheng B, et al. Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma. Int J Cancer 2001; 94:73–80. 25. Lin CL, Lo WF, Lee TH, et al. Immunization with Epstein-Barr Virus (EBV) peptide-pulsed dendritic cells induces functional CD8þ T-cell immunity and may lead to tumor regression in patients with EBV-positive nasopharyngeal carcinoma. Cancer Res 2002; 62:6952–6958. 26. Comoli P, De Palma R, Siena S, et al. Adoptive transfer of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T cells with in vitro antitumor activity boosts LMP2-specific immune response in a patient with EBV-related nasopharyngeal carcinoma. Ann Oncol 2004; 15:113–117. 27. Straathof KC, Bollard CM, Popat U, et al. Treatment of nasopharyngeal carcinoma with Epstein-Barr virus—specific T lymphocytes. Blood 2005; 105:1898–1904. 28. Bollard CM, Aguilar L, Straathof KC, et al. Cytotoxic T lymphocyte therapy for Epstein-Barr virusþ Hodgkin’s disease. J Exp Med 2004; 200:1623–1633. 29. Lucas KG, Salzman D, Garcia A, et al. Adoptive immunotherapy with allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes for recurrent, EBV-positive Hodgkin disease. Cancer 2004; 100:1892–1901. 30. Bollard CM, Straathof KC, Huls MH, et al. The generation and characterization of LMP2-specific CTLs for use as adoptive transfer from patients with relapsed EBV-positive Hodgkin disease. J Immunother 2004; 27:317–327. 31. Sherritt MA, Bharadwaj M, Burrows JM, et al. Reconstitution of the latent T-lymphocyte response to Epstein-Barr virus is coincident with long-term recovery from posttransplant lymphoma after adoptive immunotherapy. Transplantation 2003; 75(9):1556–1560. 32. Bharadwaj M, Sherritt M, Khanna R, et al. Contrasting EpsteinBarr virus-specific cytotoxic T cell responses to HLA A2-restricted epitopes in humans and HLA transgenic mice: implications for vaccine design. Vaccine 2001; 19(27):3769–3777. 33. Rickinson AB, Moss DJ. Human cytotoxic T lymphocyte responses to Epstein-Barr virus infection. Annu Rev Immunol 1997; 15:405–431. 34. Duraiswamy J, Bharadwaj M, Tellam J, et al. Induction of therapeutic T-cell responses to subdominant tumor-associated viral

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61 Herpes Simplex Vaccines David M. Koelle University of Washington, Seattle, Washington, U.S.A.

Lawrence R. Stanberry and Reuben S. Carpenter Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, New York, U.S.A.

Richard J. Whitley Department of Pediatrics, Microbiology, Medicine, and Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.

INTRODUCTION Our knowledge of the pathogenesis, natural history, treatment and molecular biology of herpes simplex virus (HSV) and its resultant infections has increased dramatically over the past decade. These advances, in part, paralleled the development of antiviral drugs that are selective and specific inhibitors of viral replication. The unequivocal establishment of the value of antiviral therapy has had a major impact on altering the severity of human disease and has major implications for long-range control of HSV infections. Recent clinical trials have demonstrated the possibility of decreasing HSV transmission between sexual partners (1). However, some clinical diseases (e.g., herpes simplex encephalitis and neonatal HSV infections) are still associated with significant mortality and morbidity in spite of antiviral therapy. Even with the rapidly evolving knowledge of the molecular biology of HSV, the development of a successful vaccine—either subunit or live attenuated—has been difficult to achieve. The development of an HSV vaccine has attracted the interest of investigators for over 100 years, but the goal of producing a licensed vaccine that protects against HSV disease has not been realized. Only now is there one promising candidate in registrational clinical trials. In large part, the unique properties of HSV—especially its ability to become latent and reactivate—and its human biology make the potential success of a vaccine more difficult to achieve than with many other viral pathogens. This chapter will review the unique problems of HSV infections and consider approaches, which have been developed historically. Further, initial success with subunit vaccines and the application of genetically engineered HSV to treatment of human disease will be described.

HISTORY Human HSV infections have been documented since ancient Greek times (2–4). Descriptions of cutaneous spreading lesions thought to be of herpetic etiology appeared in the writings of Hippocrates, as reviewed (4,5). Greek scholars defined the word ‘‘herpes’’ as ‘‘creep or crawl’’ in reference to the spreading nature of the skin lesions (2,3). The Roman scholar

Herodotus associated mouth ulcers and lip vesicles with fever and defined this association as ‘‘herpes febrilis’’ (6). Likely, many of these original observations reiterated Galen’s deduction that the appearance of such lesions was an attempt by the body to rid itself of evil humors, and, perhaps, led to the name ‘‘herpes excretins’’ (2). In any case, the fact that herpes has existed for so many centuries illustrates the host’s ability to evade immune responses and recur. However, these original descriptions of skin lesions probably bear little resemblance to later reports of the 19th and 20th centuries (6). As noted by Wildy (2), Shakespeare described recurrent labial lesions. As he wrote in Romeo and Juliet, Queen Mab, the midwife of the fairies, stated, ‘‘O’er ladies lips, who straight on kisses dream, which oft the angry Mab with blisters plagues, because their breaths with sweetmeats tainted are.’’ In the 18th century, Astruc, physician to the King of France, drew the appropriate correlation between herpetic lesions and genital infection (7). By the early 19th century, the vesicular nature of lesions associated with herpetic infections was well ascertained. However, it was not until 1893 that Vidal specifically recognized human transmission of HSV infections (2). Observations from the early 20th century brought an end to the early imprecise descriptive era of HSV infections. First, histopathologic studies described the multinucleated giant cells associated with herpesvirus infections (8). Second, the unequivocal infectious nature of HSV was recognized by Lowenstein in 1919 (9). He experimentally demonstrated that virus retrieved from the lesions of humans with HSV keratitis or the vesicles of patients with HSV labialis was transmissible, producing lesions on the rabbit cornea. These corneal lesions were similar to that encountered in humans with HSV eye infection. Furthermore, the vesicle fluid from patients with herpes zoster failed to reproduce similar dendritic lesions in the rabbit eye model. In fact, these observations were actually attributed to earlier investigations by Gru¨ter who performed virtually identical experiments around 1910 but did not report them until much later (10).

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Reports between 1920 and the early 1960s focused on the clinical and biologic manifestations of HSV as well as the natural history of human disease. During these four decades, the host range of HSV infections was expanded to include a variety of laboratory animals, chick embryos, and, ultimately, in vitro cell culture systems. Expanded animal studies demonstrated that transmission of human virus to the rabbit resulted not only in corneal disease, but also could lead to infections of either the skin or central nervous system (11), as reviewed (3,12–14). Host immune responses to HSV were reported initially in the early 1930s. The first studies were performed by Andrews and Carmichael, who defined the presence of HSV neutralizing antibodies in the serum of previously infected adults (15). Subsequently, some of these patients developed recurrent labial lesions, albeit less severe than those associated with the initial episode. This observation led to the recognition of a unique biologic property of HSV, namely the ability of these viruses to recur in the presence of humoral immunity—a characteristic known as reactivation of latent infection. Only individuals with neutralizing antibodies developed these recurrent vesicular lesions, a paradoxical finding given the classical lessons of such infectious diseases as measles and rubella whereby antibodies were typically associated with protection from subsequent episodes of disease. By the late 1930s, it was well recognized that infants with severe stomatitis, who shed a virus thought to be HSV (16), subsequently developed neutralizing antibodies during the convalescent period (17). Later in life, some of these children had recurrent lesions of the lip. The medical literature of the 1940s and 1950s was replete with descriptions of disease entities such as primary and recurrent infections of mucous membranes and skin (e.g., gingivostomatitis, herpes labialis and genitalis, herpetic whitlow or eczema herpeticum)(18), keratoconjunctivitis (19), neonatal HSV infection, visceral HSV infections of the immunocompromised host, and HSV encephalitis (20). The clinical spectrum of HSV infections subsequently was expanded to include Kaposi’s varicella-like eruption and severe and prolonged recurrent infections of the immunocompromised host. Significant laboratory advances have provided a foundation for the application of molecular biology to the study of human disease and, no less, vaccine development. These advances include, among others: (i) detection of antigenic differences between HSV-1 and HSV-2 (4,21–23); (ii) proven antiviral therapy for virtually all manifestations of HSV disease (24–34); (iii) application of restriction endonuclease technology to HSV strains to show epidemiologic relatedness (35); (iv) definition of type-specific antigens allowing the development of serologic assays that distinguish HSV-1 from HSV-2 (36,37); (v) the characterization of the replication of HSV, its resultant gene products, and the biologic properties of some of these products (38); and (vi) the attenuation of HSV through genetic engineering and the subsequent expression of foreign genes, providing technology for the development of new vaccines (39).

THE INFECTIOUS AGENT HSV, types 1 and 2, are members of a family of large DNA viruses that contain centrally located, double-stranded DNA. All of the herpesviruses have similar structural elements arranged in concentric layers (38,40,41). Other members of the human herpesvirus family include cytomegalovirus, varicella-zoster virus, Epstein–Barr virus, human herpesvirus 6, 7,

and 8 (which is also known as Kaposi sarcoma virus). The DNA of HSV has a molecular weight of approximately 100 million and encodes for about 80 polypeptides, an increasing number of which have biologic functions that are understood. The genome consists of two components, a unique long and unique short region, which can invert on themselves, allowing for the coexistence of four isomers in virus suspensions (38,41,42). The genomic arrangement of HSV-1 and HSV-2 indicates that a number of genes are collinear with reasonable, but not identical, matching of base pairs. Viral DNA is packaged inside a protein structure known as the capsid, which confers icosapentahedral symmetry to the virus. The capsid consists of 162 capsomers and is surrounded by a tightly adherent membrane known as the tegument (43). An envelope loosely surrounds the capsid and tegument, consisting of glycoproteins, lipids, and polyamines. The envelope glycoproteins are primarily responsible for the induction of humoral immune responses. The replication of HSV is characterized by the expression of three gene classes: a, [immediate-early (IE)], b (early), and g (late) genes, respectively, although there is some overlap between of each of these classes. These genes are expressed temporally and in a cascade fashion (38,44,45). Several observations are relevant as they relate to the replication of HSV for vaccine development. First, although herpesvirus genes carry transcriptional and translational signals similar to those of other DNA viruses, the mRNAs arising from the vast majority of genes are not spliced (46,47). Second, the information density is lower than that encoded in the genes of smaller viruses (38,47), permitting insertion and deletion of genes into the HSV genome without significant alteration of the genomic structure. This property provides an opportunity to genetically engineer HSV as either a vaccine or a vector for the delivery of foreign antigens (39). In this later circumstance, expression of foreign genes in HSV (i.e., cytokines) could provide an endogenous adjuvant (i.e., GM-CSF) or be useful for gene therapy. Finally, replication requires the expression of a viral coded protein, a-transinducing factor, a potentially unique target for antivirals or antigens for vaccines (38). The replication of HSV appears under the control of a genes, of which there are five. The b gene products include the enzymes necessary for viral replication, such as HSV thymidine kinase and DNA polymerase, as well as the regulatory proteins. These genes require functional a gene products for expression. The onset of expression of b gene products coincides with the decline in the rate of expression of a genes and an irreversible shut-off of host cellular macromolecular protein synthesis (44,48). This latter event equates with cell death. Structural proteins are usually of the g gene class (44,49). The g gene products are heterogeneous and are differentiated from b genes solely by the requirement for viral DNA for maximum expression of their genes. Assembly of virus begins in the nucleus with formation of empty capsids, insertion of DNA and acquisition of the envelope as the capsid buds through the inner lamella of the nuclear membrane. Further maturation of envelope glycoproteins occurs in the endoplasmic reticulum. Eleven glycoproteins have been described; these are gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM (38,50). The biologic properties of some of these glycoproteins have been identified (13,48). For example, gD is related to viral infectivity and is the most potent inducer of neutralizing antibodies; it is required for cell entry. gB is required for infectivity. These two glycoproteins have been

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utilized extensively in subunit vaccines, as discussed below. gC binds to the C3b component of complement while gE binds to the Fc portion of IgG. In addition, it appears as though a deletion in gC enhances viral pathogenicity (51). gG provides antigenic specificity to HSV and, therefore, results in an antibody response that allows for the distinction between HSV-1 and HSV-2 (36). gI interacts with gE to form an Fc receptor (52,53). The importance of these glycoproteins for vaccine development cannot be overemphasized. Considerable antigenic cross-reactivity exists between the HSV-1 and HSV-2.

HOST-VIRUS INTERACTION In considering the development of vaccines directed against HSV, some understanding of the induced pathology and its implications on the modulation of the biology of human disease is relevant. The pathogenesis of HSV infections can best be understood through knowledge of the events of replication and establishment of latency in both animal models and humans. Infection is initiated by contact of the virus with mucosal surfaces or abraded skin. The fundamental principle of the pathogenesis of human infections is that transmission occurs by intimate personal contact, resulting in viral replication at the mucosal surfaces of initial infection. With viral replication at the site of infection, either an intact virion or, more simply, the naked capsid enters the nerve termini and is carried by retrograde axonal flow to the dorsal root ganglia where, after several cycles of viral replication, latency is established (54). These events have been demonstrated in a variety of animal models, as reviewed (55). After latency is established, reactivation can occur with a proper provocative stimulus (i.e., stress, menstruation, fever, exposure to ultraviolet light, etc.), and virus is transported anterograde down the axon to replicate at mucocutaneous sites, appearing as skin vesicles or mucosal ulcers. Viral replication can lead to systemic disease such as disseminated neonatal HSV infection with multiorgan involvement, or, very rarely, multiorgan disease of pregnancy and, infrequently, dissemination in severely immunosuppressed patients. Presumably, widespread organ involvement is the consequence of viremia in a host not capable of limiting replication to mucosal surfaces. Infection with HSV-1 is commonly transmitted to the oropharynx by direct contact of a susceptible individual with infected secretions (such as virus contained in labial vesicular fluid) (56–58). Thus, initial replication of virus occurs in the oropharyngeal mucosa; the trigeminal ganglion becomes colonized and harbors latent virus. Acquisition of HSV-2 infection is usually the consequence of transmission via genital routes, although genital to oral transmission can occur. Under these circumstances, virus replicates in the vaginal tract or on penile skin sites with colonization of the sacral ganglia. For uncertain reasons, HSV-1 infection, when symptomatic, recurs more frequently in the oropharyngeal area, and HSV-2, when symptomatic, recurs more frequently in the genital area (59). The epidemiology and clinical characteristics of primary infection are distinctly different from that associated with recurrent infection. This subject has been reviewed extensively (5,12,13,60,61). During primary infection of the genital tract or oropharynx, viral replication persists for nearly two weeks and disease for three weeks. After the establishment of latency, reactivation of HSV, either symptomatic or asymptomatic, is known as recurrent infection. During recurrent symptomatic

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infection, viral replication persists 24 to 48 hours and total disease lasts seven to ten days. Symptomatic reactivation leads to recurrent skin vesicular lesions such as HSV labialis or recurrent HSV genitalis. Individuals with preexisting antibodies to one type of HSV can experience a first infection with the opposite virus type at the same or different site. This occurrence is known as an initial infection rather than primary. An example of an initial infection would be those individuals who have preexisting HSV-1 antibodies, acquired after HSV gingivostomatitis, who then acquire a genital HSV-2 infection. The natural history of an initial infection more closely resembles recurrent infection. Preexisting antibodies to HSV-1 appear to have an ameliorative effect on disease associated with HSV-2 infection (62). Both initial and primary infections have also been named first-episode infection. Importantly, shedding of virus can be either symptomatic or asymptomatic. Indeed, transmission occurs more frequently during periods of asymptomatic shedding. Reinfection with a same strain of HSV can occur, although the frequency of such events is unknown (63,64). This occurrence is defined as exogenous reinfection. Ideally, vaccines directed against HSV should prevent infection as well as disease. However, it is unlikely that such a vaccine will uniformly confer protection from infection, as discussed below in the context of a recently completely clinical trial. Thus, a vaccine should significantly alter the clinical manifestations of primary disease and, perhaps, the frequency and severity of recurrent infection, since there is a direct relationship between the number of infected ganglionic neurons and ‘‘recurrence’’ in mice (65) and guinea pigs (66). In so doing, it is anticipated that transmission of infection will be decreased.

ANIMAL MODELS OF HSV VACCINE DEVELOPMENT Numerous animal models have been used to study the pathogenesis of HSV infections, especially latency, antiviral therapeutics, and vaccines. Candidate HSV vaccines have been evaluated in rodents (mice, guinea pigs, rabbits, etc.) and subhuman primates. Selection of the animal model system for vaccine evaluation is relevant. The animal species, virus type, route of infection, state of immune competence, and specific viral challenge strain all influence disease pathogenesis and, synonymously, the evaluation of a vaccine. Disease progression alone can be selectively modulated by the specific strain of animal selected. The route and site of inoculation of virus become especially important in distinguishing one disease state from another and its prevention. When HSV-1 is inoculated by the ocular route, encephalitis (virulence) and/or latency ensue, as reviewed (67). The eye route of infection has been best utilized to study latency. Endpoints of virulence and latency, however, have distinct differences for predicting human disease. Following ocular inoculation after corneal scarification, replication of virus in the eye peaks within 48 hours and declines over the next 6 days. Virus appears in the trigeminal ganglia approximately one day after inoculation with peak replication occurring between four and six days. Subsequently, if the inoculum of virus is large enough and/or a virulent strain of virus is used, brain infection follows invasion of the trigeminal ganglia. Fortunately, while the trigeminal ganglion is routinely infected following human eye infection (68,69), subsequent invasive brain

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infection is an exceedingly uncommon event, if ever having occurred. This model has been used by Hendricks et al. (70) and confirmed by other groups (71) to demonstrate a key role for HSV-specific CD8 T cells in the immune control of HSV. When mice are inoculated in the eye with an HSV-1 strain and amount leading to recovery and latent trigeminal ganglia infection, these investigators have found that high levels of HSV-specific T cells persistently localize to the ganglia, where they participate in virus control through the secretion of interferon (IFN)-g. These findings were recently extended to humans, with autopsy studies showing that HSV-1-reactive CD8 T cells with IFN and cytotoxic effector functions are specifically localized to infected ganglia (72). Mice, particularly the hairless mouse (73), rabbits and guinea pigs with abraded or punctured skin have been used to study virus replication and pathogenesis of HSV-1, and to evaluate both antiviral therapies and vaccines. While in each model, the nature of the lesions, their duration and histopathology parallel human infection, there are notable differences, particularly the lack of recurrent lesions similar to those seen in people. Type 2 genital infections have been studied best in the guinea pig model. Intravaginal inoculation of HSV into female Hartley guinea pigs appears the most predictive of all the animal models for human disease (74). These animals tend to suffer from continual recurrences of lesions; however, retrieval of HSV from these lesions is most variable. Recently, PCR of recurrent genital lesions in the guinea pig model has added microbiologic certainty to the evaluation of vaccines and drugs in this model (75). Models of life-threatening disease have been developed for both HSV-1 and HSV-2. Intranasal inoculation of HSV-1 or HSV-2 in young (3 weeks of age) Balb/c mice leads to central nervous system and visceral (usually lung) disease, which may be predictive for neonatal HSV infection. However, inoculation of older mice with similar quantities of either virus may lead to no evidence of disease or, if so, encephalitis but certainly not overwhelming multiorgan disease. Direct intracerebral inoculation of virus is an unnatural route of infection even for the study of either antiviral therapeutics or vaccine efficacy. Accurate and predictive models of human HSV encephalitis have been described in a rabbit model (76–78). Virus is inoculated directly into the olfactory bulb or on abraded nasal epithelium over nerves from the olfactory bulb; it can be traced along the olfactory tract to the anterior-frontal region of the rabbit brain where it causes focal infection, as compared with the diffuse pancortical infection, which follows infection by the murine ocular route. The region of the rabbit brain involved correlates with the temporal lobe of humans. Immunosuppression following subclinical infection can result in focal reactivation (78). Primate models have been utilized to study vaccines. These animals have been thought to more closely approximate that which is encountered in humans; however, the disease pathogenesis varies for each primate species. Specifically, the Aotus monkey (Aotus trivirgatus or nancymani) is exquisitely sensitive to HSV and, therefore, serves as a useful model for assessment of attenuated live vaccines but is, perhaps, less amenable to protection studies. The demonstration of safety in the Aotus model provides confidence of safety before introducing such a vaccine into humans (79). In contrast, the owl monkey is far less susceptible to HSV, and therefore may more closely resemble humans.

HOST IMMUNE RESPONSES Primary HSV Infection Local control mechanisms of viral spread aim to neutralize the infectious agent and lead to viral clearance. Following primary HSV infection, the initial, local immunologic responses involve both nonspecific defense mechanisms, namely IFNs-a and IFNs-b, activated natural killer (NK) cells and macrophages, as well as HSV-specific responses, such as cytotoxic T cells (CTLs) (80). In response to a viral infection, the initial cellular response is synthesis and secretion of type I IFNs (a and b). IFNs induce an antiviral state in infected and surrounding cells. The antiviral activity is modulated in part by IFN-mediated activation of cellular enzymes such as 20 -50 oligoadenylate synthetase (20 -50 AS) and double-stranded RNA-dependent protein kinase, as well as intracellular signaling molecules through the activation of the JAK/STAT kinase pathway. More specific to HSV infection, IFN-a appears to inhibit IE gene expression (81). Thus, the antiviral mechanism directly affects transactivation of the IE responsive element necessary for synthesis of viral proteins. In addition to antiviral activity, IFNs are potent immunomodulators. As such, they mediate macrophage and NK-cell activation, activate CTLs, induce major histocompatibility complex (MHC) class I and MHC class II antigens, stimulate cytokine secretion and induce local inflammation. IFN-g may aid the control of HSV infection. Evidence that gd T cells, NK cells, CD4þ T cells and possibly neurons produce IFN-g and TNF in response to HSV infection in the nervous system has been reported. IFN-g down regulates priming of CD4þ Th2 cells, which are responsible for inducing Ig isotype B cell switching from IgA to IgG, thereby exerting a major effect on humoral immune responses (82). NK cells lyse pathogen-infected cells before virus-specific T-cell immunity is generated and constitute first line defense against infection. In vitro and in vivo experiments have demonstrated that NK cells protect from HSV challenge in a murine model (83). Severe herpetic disease has correlated with low in vitro NK activity in newborns, as well as in a patient lacking NK cells (84). Other mononuclear cells, such as macrophages, are recruited to the site of infection and, upon activation, release immune cell mediators such as TNF and interleukins. Macrophages play a major role in mediating antibodydependent cellular toxicity for viral clearance and antigen presentation (85). An important aspect of immune responses to HSV infections is the maturation of dendritic cells (DCs) at the site of infection. Mobile DC travel from mucosal or skin areas of infection and prime antigen-specific, naive T cells in draining lymph nodes (DLN). Recent work using HSV-1 footpad infection and fractionating DC subpopulations in the DLN show that classic, CD8aþ dermal DC, rather than specialized epidermal Langerhan’s cells (LC), are able to prime naı¨ve CD8þ T cells (86). In the case of vaginal HSV-2 infection, dermal DC rather than LC again seem to be the physiologically active cell population in a similar DLN investigation (87). LC are certainly able to present HSV antigens to memory HSV-specific T cells, and may participate in primary or recurrent immune reactions. The recently described plasmacytoid DC (pDC) population reacts to HSV by producing copious amounts of IFN-a (88). pDC are recruited to sites of infection, participate in viral clearance, and express relevant Toll-like receptors (TLRs) include TLR 7, 8, and 9, the later having been shown to recognize HSV (89,90). Of interest, low pDC number or poor

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pDC reactivity with HSV is associated with severe HSV infection in humans (91,92). As infection progresses, virus-specific immune responses are detected. On days 4 and 5 postinfection, HSV-specific CD4þ Th1 lymphocytes are detected in genital lymph nodes and in smaller numbers in peripheral blood; they can subsequently be found in the genital mucosa (93). CD8 responses also occur quite quickly in the mouse (94); relevant human studies have not yet been reported. Humoral immune responses rapidly follow initial HSV infection. The predominant mucosal antibodies are of the IgA isotype, being secreted by plasma cells. These antibodies can be detected as early as day 3 following infection, peaking within the first six weeks after disease onset, and are followed by appearance of IgG1 and IgG3 subclasses of antibodies, which are typically found following viral infections. HSVspecific IgA antibodies are present for at least six weeks, gradually decreasing to undetectable levels. IgM-secreting B cells have also been detected in secretions of the female genital mucosa (95). Shorter periods of viral shedding in women with primary genital herpes have been positively correlated with presence of secretory IgA in vaginal secretions (83).

Recurrent HSV Infection Although immunosuppression enhances the frequency of reactivation, there is no proof that the immune system exerts any influence on reactivation at the level of the ganglia (96). Immunosuppression enhances the detection of HSV reactivation in the periphery. It is not clear if this is due to increased ganglionic reactivation, or failure of control mechanisms in the skin to contain virus being delivered down the axon. Newer data show that HSV-specific CD8 and CD4 T cells persistently infiltrate latently infected trigeminal ganglia in mice and humans (70,72). These cells appear to act via IFN-g (97). They have cytolytic activity, but neuron loss is not seen clinically, and inhibitory receptor-ligand pairings can be documented in the ganglia that may modulate their cytotoxic activity (71). In the periphery, HSV2 specific CD4 and CD8 T cells localize to sites of recurrent HSV-2 infection and to the cervix (98,99). Using in situ staining, HSV-2specific CD8 CTL have been shown to persist at the epidermal/ dermal junction adjacent to sensory nerve endings (100). Damage to these cell populations in immune compromised persons may

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lead to increased HSV replication either centrally or peripherally. Repeated subclinical episodes of HSV excretion may be a source of antigenic stimulation leading to long-term HSV-specific immune memory (85). In recurrent HSV-2 infections, NK and HSV-specific CD4þ cells are detected earlier than CD8þ cells in genital lesions (82). CD4þ T cells, and more recently, CD8þ T cells have been highlighted as major mediators of viral clearance from mucocutaneous lesions in recurrent episodes (80,98,101). Low IFN-g titers in vesicle fluid have been associated with a shorter time to the next recurrence in patients with frequent recurrences. T-cell proliferation is decreased in these patients in comparison with patients with less frequent recurrences (85). In as much as the involvement of cytokines has been studied, IFN-g has been reported to have a role in viral clearance from mucocutaneous sites, whereas altered cytokine production appears to correlate with recurrence (102). As with primary HSV infection, a shorter duration of viral shedding occurs in women with recurrent genital herpes who have detectable secretory IgA in vaginal secretions (83). IgA, IgG1 and IgG3 antibodies have been found in the sera of all patients with recurrent HSV-2 episodes, while IgM and IgG4 antibodies were detected in 70% to 80% of these patients. However, there does not appear to be clear correlation between humoral immune responses and disease prognosis (103). Table 1 summarizes the immune responses to primary versus recurrent herpes infection.

Persistence of Immune Responses The host’s immune responses persist and partially control HSV disease; recurrent episodes are generally less severe and of shorter duration over the years, perhaps because of progressive enhancement of long-term immunity (104). Furthermore, some degree of cross-protection exists between HSV-1 and HSV-2, as noted above. Additionally, newborns are partially protected by maternal antibodies (105). Finally, HSV-specific T-cell infiltrates are detected in herpetic lesions during early disease resolution (80). Studies indicate that persistent cell-mediated immune responses are more important than humoral immune responses in the resolution of HSV disease (106). NK cells, macrophages and T-lymphocytes as well as cytokines such as IFNs-a and IFNs-g, IL-2 and IL-12 all have central roles in resolving HSV disease (107). HSV-specific CD4þ and CD8þ cells are detected

Table 1 Summarizes the Immune Responses to Primary Versus Recurrent Herpes Infection Primary infection Local response Early nonspecific response IFN-a and IFN-b, NK cells, macrophages (3 to 4 hr after infection, appearance of viral glycoproteins as targets for ADCC). Mucosal dendritic cells are MHC class II positive antigen-presenting cells, acting as antigen-presenting cells. HSV infection induces maturation of these cells that produce high levels of type I IFN. From days 4 to 5: HSV-specific CD4þ Th1 appear in genital lymph nodes, then in the genital mucosa. Cervical immunoglobulin A (IgA) antibodies to several HSV-2 glycoproteins and IgG responses follow. Systemic response From 2 wk: detection of IgG to HSV glycoproteins in primary HSV-2 infected patients. IgA and IgG types of HSV-specific antibodies are maintained for at least 6 wk. IgG responses increase in the first year after primary infection and are detected in all genital herpes patients. Recurrent Infection Local response 12 to 24 hr after appearance of recurrent lesions: HSV-specific CD4þ and CD8þ cells, as well as macrophages are detected, with early predominance of CD4þ cells. Systemic response Frequent subclinical reactivations may maintain relatively high frequencies of HSV-specific memory T cells. CD8þ cells have been recently proposed as being a critical component in recurrent disease resolution. Abbreviations: IFN, interferon; NK, natural killer; HSV, herpes simplex virus; ADCC, antibody-dependent cellular cytotoxicity; MHC, major histocompatibility complex. Source: From Ref. 314.

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in lesions from recurrent episodes, indicating their potential role in controlling HSV disease (80,98). By contrast, agammaglobulinemic patients do not experience more severe or more frequent herpetic recurrences than the general population (108). Furthermore, several vaccine trials have demonstrated that the presence of neutralizing antibodies to HSV glycoproteins does not provide protection against HSV infection or disease.

Correlation of Immune Responses with Disease Vaccine development, in general, requires the correlation of disease with host immune responses. The development of a vaccine to prevent HSV infections is no exception. Nevertheless, efforts to precisely incriminate that arm of the host response responsible for disease have remained elusive. Humoral immunity to HSV infection has been evaluated exhaustively in disease pathogenesis. Polyclonal antibodies have been used to alter disease lethality, particularly in the newborn mouse or to limit progression of both neurologic and ocular disease (109–113). Monoclonal antibodies to selected specific infected cell polypeptides, especially the envelope glycoproteins gB and gD, confer protection from lethality (114–116). Importantly, gD2 is a known target of neutralizing antibodies, antibody-dependent cell-mediated cytotoxicity and CD4 and CD8 T cell–mediated responses (117–120). As a consequence, this antigen has been a prime component of subunit vaccines. Efforts to correlate the frequency recurrences with immune responses have failed to identify any specific humoral response to specified polypeptides (106,121,122). Thus, further efforts focused, in large part, on cell-mediated immune responses. As noted, lymphocyte blastogenic responses are demonstrable within four to six weeks after the onset of infection and sometimes as early as two weeks (123–130). These responses are typically medicated by CD4 T cells. With recurrences, boosts in blastogenic responses occur; however, these responses, as after primary infection, decrease with time. Nonspecific blastogenic responses do not correlate with a history of recurrences. HSV-1 and HSV-2 are cross-reactive in these assays at the whole virus levels, although individual T-cell clonotypic responses can be either type-common or type-specific (131). Lymphokine production has been incriminated in the pathogenesis of frequently recurrent genital and labial HSV infection. Notably, several investigators have recognized a decrease in both IFN-g production and natural killer cells during disease prodrome (132–134). Nevertheless, there are no reproducible data from selected populations to confirm these observations. The relevance of lymphokine expression in vaccine development can be assessed only in prospective field trials. Host response of the newborn to HSV must be defined separately from that of older individuals. Immaturity of host defense mechanisms is a cause of the increased severity of some infectious agents in the fetus and the newborn. Factors that must be considered in defining host response of the newborn include also the mode of transmission of the agent (viremia vs. mucocutaneous infection without blood-borne spread), time of acquisition of infection and the potential of increased virulence of certain strains, although this last point remains purely speculative. Two broad issues are of relevance; these are protection of the fetus by transplacental antibodies and definition of host responses of the newborn. Transplacentally acquired neutralizing antibodies either prevent or ameliorate infection in exposed newborns as do antibody-dependent cellmediated cytotoxicity (135–137). Importantly, preexisting

antibodies, indicative of prior infection, significantly decrease the transmission of infection from pregnant women to their offspring (138), contributing to the rationale for the development of a HSV vaccine. Humoral IgG and IgM responses have been well characterized. Infected newborns produce IgM antibodies specific for HSV, as detected by immunofluorescence, within the first three weeks of infection. These antibodies increase rapidly in titer during the first two to three months, and they may be detectable for as long as one year after infection. The most reactive immunodeterminants are the surface viral glycoproteins, particularly gD. Humoral antibody responses have been studied using contemporary immunoblot technology and the patterns of response are similar to those encountered in adults with primary infection (130,139). The quantity of neutralizing antibodies is lower in babies with disseminated infection (130,136). Cellular immunity has been considered to be important in the host response of the newborn. The T-lymphocyte proliferative response to HSV infections is delayed in newborns compared with older individuals (130). Most infants studied in a recent evaluation had no detectable T-lymphocyte responses to HSV two to four weeks after the onset of clinical symptoms (125,130,140). The correlation between these delayed responses may be of significance in evaluating outcome to neonatal HSV infection. Specifically, if the response to T-lymphocyte antigens in children who have disease localized to the skin, eye or mouth at the onset of disease is significantly delayed, disease progression may occur at a much higher frequency than babies with a more appropriate response (130,141). Infected newborns have decreased production of IFN-a in response to HSV when compared with adults with primary HSV infection (130). The importance of the IFN generation on the maturation of host responses, particularly the elicitation of NK-cell responses, remains to be defined (142,143). Lymphocytes from infected babies have decreased responses to IFN-g during the first month of life (130,143,144). These data taken together would indicate that the newborn has a poorer immune response than older children and adults. Antibodies plus complement and antibodies mixed with killer lymphocytes, monocytes, macrophages or polymorphonuclear leukocytes will lyse HSV-infected cells in vitro (145). Antibody-dependent cell-mediated cytotoxicity has been demonstrated to be an important component of the development of host immunity to infection (146). However, the total population of killer lymphocytes of the newborn seems to be lower than that found in older individuals and monocytes and macrophages of newborns are not as active as those of adults (147–152). These findings are supported by animal model data.

CRITERIA FOR EVALUATION OF HUMAN HERPESVIRUS VACCINES The development of an efficacious HSV vaccine is much needed, as best defined by disease burden. In the United States alone, over 100 million individuals are infected by HSV-1 and at least 40 to 60 million individuals have been infected by HSV-2 (153). Annually, a minimum of 2500 cases of neonatal herpes and 3000 cases of herpes simplex encephalitis results in significant morbidity and mortality in spite of efficacious antiviral therapy. Furthermore, because HSV results in genital ulcerative disease, the risk of acquisition of human immunodeficiency virus (HIV) is significantly increased (154–157).

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An ideal vaccine should induce immune responses adequate to prevent infection. If primary infection was prevented, the colonization of the sensory ganglia would not occur and, therefore, no source of virus for either subsequent recurrences or transmission would exist. No one knows today if such ideal objectives can be met. In animal models, true ‘‘sterilizing immunity’’ that prevents detectable wild-type virulent virus from becoming established in a latent state can be achieved, but typically only by live attenuated vaccines that themselves become latent, but may be less efficient than wild-type viruses for reactivation from latency (158,159). In fact, it is open to discussion whether the criteria of success for candidate vaccines should be one or any combination of the following effects: abrogation or mitigation of primary clinical episodes, prevention of the colonization of the ganglia, suppression or reduction of the frequency and/or of the severity of recurrences, reduction of the shedding (duration and/or quantity) of the virus during primary and/or recurrent episodes, reduction of asymptomatic shedding (frequency, duration, quantity) and/or prevention of person to person transmission (either vertical or horizontal). These issues must be weighed in the context of the age of the target population and the duration of the desired results. Arguably, fundamental to a successful vaccine is the last point—namely, its ability to prevent person-to-person transmission to interrupt the ongoing HSV epidemic. To illustrate the difficulty of achieving such an end, overt clinical recurrences may only be apparent in approximately 20% of HSV-infected individuals, resulting in a large reservoir of unknowingly infected individuals who may intermittently excrete virus in absence of symptoms and transmit it to intimate partners (160). Further complicating the issue, HSV DNA has been detected by polymerase chain reaction (PCR) in the genital secretions of women without either lesions or evidence of infectious virus (161), usually threefold more frequently than infectious virus. Subsequent person-to-person transmission has been documented from infected mothers to their newborns. The persistent detection of HSV DNA implies that HSV is a more chronic infection than previously thought and a more difficult vaccine target. Prospective clinical trials, a time-consuming and expensive exercise, will be required to appropriately define the true utility of an HSV vaccine. For these reasons, it is of the utmost importance to determine which factors are protective against HSV infection (humoral vs. cell-mediated immunity, local immunity vs. systemic immunity, antibody-dependent cellmediated cytotoxicity vs. CTLs) so that markers can be developed to expedite the evaluation of vaccines in humans (79). The rationale for an HSV vaccine is threefold. First, exogenous reinfection is exceedingly uncommon in the immune competent host (although it has been documented) (162). Second, many more individuals are infected by HSV than experience either clinical recurrences or shed virus. Finally, transplacental antibodies significantly decrease the risk of infection in the newborn exposed to HSV at the time of delivery. Taken together, these facts strongly suggest that a properly designed vaccine could be efficacious.

HERPES SIMPLEX VIRUS VACCINE DEVELOPMENT Attempts to develop a HSV vaccine have been tested for over a century. While all of these approaches will be reviewed, in the 21st century, the two of the more promising HSV vaccines

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represent entirely different theoretical approaches. The first is based on either microorganisms or cell lines producing gB or gD2 for use as subunit vaccines in combination with an adjuvant. The second is based on genetically engineering the virus so as to yield either a live, attenuated vaccine from which putative neurovirulence and immune evasion sequences have been removed or to produce a vaccine virus, which is only capable of a single round of replication. Each of these approaches has been evaluated extensively in animal model studies and to varying extents in human investigations. Extrapolating protection from animal model systems to humans has not been possible because there are no markers of protection comparable to neutralizing antibodies for other viral diseases (163–170). While initial HSV vaccine efforts were oriented toward the prevention of recurrent infections and, therefore, were considered therapeutic vaccines, more recent efforts have been devoted to the prevention of infection or disease following exposure to an infected partner. This review will concentrate on prophylactic vaccines. The approaches to HSV vaccine development include the utilization of: (i) wild-type virus, (ii) inactivated or killed virus, (iii) subunit vaccines, and (iv) genetically engineered vaccines.

Wild-Type Virus Numerous clinicians attempted to alter the pattern of recurrences by inoculation of autologous virus, of virus from another infected individual, or, in one set of experiments, of virus recovered from an experimentally infected rabbit (171–173). The consequences were obvious with lesions appearing at the site of inoculation in as many as 40% to 80% of volunteers (174–176). In spite of the appearance of lesions and the evaluation of only a very limited number of patients, the efficacy of such an approach was reported in the literature. Furthermore, these studies failed to utilize controls (176–178). In some cases, inoculation led to recurrences (172,173,179,180). In large part, live viruses were abandoned on the grounds that many patients did not develop lesions at the site of inoculation and, therefore, it was not perceived that the patient had an ‘‘adequate take’’(181–183). At the present time, inoculation of either autologous or heterologous virus is unacceptable.

Inactivated (or Killed) Virus Killed virus vaccines have been studied in a variety of animal model systems, often with good results, as reviewed (184–186). Unfortunately, when these vaccines were administered to HSV-infected individuals to alleviate recurrences, most studies failed to include an appropriate control group. Under such circumstances, significant bias was introduced since patients may experience 30% to 70% decrease in the frequency of recurrences as well as improvement in severity, simply from having received placebo (187–192). The initial inactivated vaccines were derived from phenol treated infected animal tissues (181–183). Because of the possibility that administration of animal proteins might lead to demyelination, these vaccines did not attract much biomedical attention. Instead, ultraviolet light inactivation of purified virus derived from tissue culture replaced phenol inactivation. Over the past two decades, numerous reports in the literature suggested either the success or failure of these approaches. As reviewed (185), viral antigen obtained from amniotic or allantoic fluid, chorioallantoic membranes, chick cell cultures, sheep kidney cells, rabbit kidney cells and inactivated either by

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formalin, ultraviolet light or heat led to a series of vaccine studies in thousands of patients (187,193–216). With one exception (193), each of these studies reported significant improvement in as many as 60% to 80% of patients (207–209,215). As these studies progressed several important observations were made. First, despite repeated inoculations, antibody titers (as measured by neutralization or complement fixation) remained unchanged in the majority of patients (196,203,217) or only demonstrated slight increases (194,214). Second, while these efforts reported few side effects, some authors noted concern that, in patients with keratitis, autoimmune phenomena might make the herpetic disease worse (204,212,218–220). Placebo-controlled studies utilizing inactivated vaccines were few, as reviewed (191,220–222). The results were widely discrepant, even when the same vaccine was utilized, a finding most discouraging. A conclusion from these investigations was that there may be some initial benefit for patients with recurrent infection; however, long-term benefit could not be established. The only prospective study of prevention of HSV infections by vaccination was performed by Anderson et al. in children of an orphanage (222). In this study ten children received vaccine and ten received placebo; yet, HSV stomatitis developed in an equal number of patients on long-term follow-up.

Subunit Vaccines Subunit vaccines evolved out of attempts to remove viral DNA and eliminate the potential for cellular transformation, to enhance antigenic concentration and induce stronger immunity and, finally, to exclude any possibility of residual live virus contamination (223). Available subunit vaccines have been prepared from a variety of methods combining antigen extraction from infected cell lysates by detergent and subsequent purification. The immunogenicity of vaccines derived from all of the envelope glycoproteins, free of viral DNA, has been demonstrated in animals (61,224–226). The results of studies in humans are conflicting. While one vaccine was reported to decrease recurrences in infected patients, the study design did not employ a placebo control (227); thus, no conclusions of efficacy can be drawn. Vaccination with envelope glycoproteins does not protect uninfected sexual partners of individuals with genital HSV infection (228,229). More recently specific subunit vaccines have arisen out of the cloning of specific glycoproteins in either yeast or Chinese hamster ovary cell systems (189,230,231), as well as by other methods (232–234). Subunit vaccines have been studied in a variety of animal models including mice (235–241); guinea pigs (237,242–246), and rabbits (247). Neutralizing antibodies can be detected in these vaccinated animals in varying amounts, and in some systems binding antibodies as well (248). In these systems the quantity of neutralizing antibody correlated with the degree of protection upon challenge. Challenges in the experimental models have been studied in mice (235– 238,241,248–250), rabbits (247,251–253), and guinea pigs(242– 244,253). Each of these systems utilized a variety of different routes of challenge as well as dosages. Challenge included skin, lip abrasion, intravaginal inoculation, intradermal ear pinna inoculation, intradermal injection, footpad challenge, intraperitoneal, ocular, or subcutaneous. Thus, interpretation of these results is extremely difficult. While there are many conflicting animal model studies, in general, the subunit vaccines elicited a degree of protection as evidenced by amelioration of morbidity and reduction in mortality in the immunized animals.

Nevertheless, several injections were required to induce protection and must include adjuvant as well. The necessity for an appropriate adjuvant has been recently emphasized (254,255). Protection in the rodent is significantly easier than in higher primate species. This may be especially the case since the HSV is not indigenous to rodent species and, thus, protection studies may be totally irrelevant when evaluating human responses. Vaccination of primates, specifically rhesus monkeys (247), chimpanzee (224,247), and cebus monkeys (250) can induce neutralizing antibodies, leading to an anamnestic response following subsequent injection months later. The significance of the protection in these animals remains unclear for human experimentation. Subunit vaccines have been evaluated in humans. Both HSV-1 and HSV-2 antigens have been prepared in human diploid cells, Chinese hamster ovary cells and chicken embryo fibroblasts for vaccine purposes. Several studies have reported evidence of improvement (227,256,257). Other studies suggested very little benefit (224,228,229). Several human subunit vaccine trials have now been completed. One of the earliest human vaccine experience was with an early Merck (250) glycoprotein envelope subunit vaccine (228,229,258). This vaccine was produced by purification of the envelope glycoproteins from infected cells. In a phase IIA study, carried out in sexual partners of patients known to have genital herpes, the number of individuals developing herpetic infection was nearly equal between placebo and vaccine recipients; thus, vaccination failed to provide any benefit at all. There were, however, issues related to the immunogenicity of the vaccine as it induced ELISA antibody titers to HSV-2 glycoproteins D (gD2) and B (gB2) that were only 10% and 5%, respectively, of titers found in persons with recurrent genital HSV-2 infection (61). More recently a series of clinical trials have evaluated the Chiron Corporation gB2 and gD2 and GlaxoSmithKline gD2 subunit vaccines in humans. These vaccines incorporated either a single or both glycoproteins with adjuvants unique to each company. From a developmental perspective, important lessons were learned. Extensive rodent experiments, utilizing the guinea pig and murine genital herpes models demonstrated that either combined gB2 and gD2 or gD2, with Freund’s adjuvant, completely protected against both primary and spontaneous recurrent disease following intravaginal viral inoculation, but did not protect the animals against infection as measured by detection of HSV-2 replication in genital secretions following virus challenge (243). However, complete Freund’s adjuvant is not acceptable for human administration. Thus, alternative adjuvants were explored, including Chiron Corporation MF59 and AS04 (50 mg 3-O-desacyl-40 -monophosphoryl lipid A (259) and 500 mg aluminum hydroxide), a proprietary GlaxoSmithKline adjuvant. Both afforded a high level of protection from HSV disease (231,254). An important finding from these preclinical studies was that the quantity of neutralizing antibody elicited by immunization and the total HSV antibody titer (as measured by ELISA) were higher after vaccination than following natural infection, and furthermore, that these antibody titers correlated with protection from disease (243,260). Both the Chiron and the GlaxoSmithKline vaccines were evaluated in large phase III studies, but the two companies chose critically different primary outcome measures. The primary outcome measure for the Chiron studies was prevention of infection as measured by seroconversion to HSV antigens not

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contained in the vaccine. The primary outcome for the GlaxoSmithKline studies was prevention of virologically confirmed symptomatic genital herpes (102). The decision of GlaxoSmithKline to select prevention of disease as the primary outcome measure was in part predicated on animal studies that showed the vaccine could afford good protection against disease, but only limited protection against infection (75). Data from the largest series of vaccinated individuals with the Chiron Corporation construct demonstrated that the vaccine induced antibody titers that exceeded those found in individuals who had sexually acquired HSV-2 infection. However, the vaccine failed to provide significant long-term prevention of infection in susceptible sexual partners, although initial benefit was apparent for the first five months (261). In this trial, there was a 50% reduction in the rate of infection among HSV seronegative women during this short window. The overall efficacy of the vaccine for one year following a six-month vaccination period was 9%. The vaccine had no apparent effect on the frequency of recurrences amongst vaccine recipients who became infected (262). No further vaccine studies are planned for this construct. Of note, the adjuvant for these studies was MF59, a potent inducer of Th-2 responses. Another series of clinical trials involving 2714 partners of patients with HSV-2 genital herpes assessed the safety, immunogenicity and efficacy of the GlaxoSmithKline gD2 vaccine (263). Here, the proprietary adjuvant, AS04, contained alum plus monophosphoryl lipid A, a potent inducer of Th-1 responses (264) and the vaccine was shown to induce robust humoral and cellular immunity. The safety and immunogenicity of the vaccine has been extensively assessed in nearly 5000 volunteers and has been shown to be generally safe, but causes more local reactions such as soreness compared with controls. The vaccine was shown to induced titers of HSV gDspecific antibody that were higher than those observed in patients who had sexually acquired genital HSV-2 infection (265). In these studies, women who were seronegative for both HSV-1 and HSV-2 were significantly protected from disease (72% efficacy; p = 0.01–0.02) and there was a trend toward protection against infection (43% efficacy; p = 0.06–0.07). However, in individuals seropositive for HSV-1, irrespective of sex, and seronegative men, no significant clinical benefit could be demonstrated. Thus, the two phase III efficacy studies showed that the GlaxoSmithKline candidate genital herpes vaccine (gDAlum/MPL) is effective in preventing HSV-1 or HSV-2 genital herpes disease in a subset of volunteers, that is, women who were HSV-1 and HSV-2 seronegative (HSV1/2) prior to vaccination. However, these studies were neither designed nor powered to assess efficacy exclusively in HSV1/2 women, and therefore did not meet their primary endpoints of overall efficacy. Consequently, a double-blind, randomized, placebo-controlled phase III study sponsored by the National Institutes of Health and GlaxoSmithKline was initiated in late 2002 to evaluate the prophylactic efficacy of gD/Alum/MPL vaccine (266). The study involves 7550 HSV1/2 sexually active women ages 18 to 30 who will be followed for 20 months.

Live Vaccines Live vaccines, in general, are considered more immunogenic, but have increased safety concerns, compared with killed or subunit vaccines. This is because they are more likely to induce a broad range of immune responses to the expressed gene products and, therefore, provide a high level of protection as

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has been the case with numerous viral pathogens such as measles, mumps, and rubella. Furthermore, since these vaccines replicate in the recipient, the resulting immunity should be longer lasting. Moreover, they usually require smaller doses of antigen and, therefore, should be more economical. Several approaches to live virus vaccines have been attempted. These include: HSV mutants, heterologous herpesviruses, antigens expressed in non-HSV viral vectors, and genetically engineered viruses. Herpes Simplex Virus Mutants For another a herpesvirus, varicella-zoster virus, tissue culture attenuation has been used to produce less virulent virus strains that are suitable for use as vaccine viruses (e.g., OKA varicella strain). It was recognized very early in biologic laboratory investigations that virulence varied significantly among wild-type HSV isolates. Conceivably, one could use the least virulent wild-type HSV as a vaccine, but reversion from nonpathogenic to pathogenic strains easily occurs following serial passages either in cell culture or animal hosts. This lack of genetic stability is unacceptable for potential human vaccines (260,267). Heterologous Herpesvirus Vaccines While considered for other herpesvirus infections, such as Marek’s disease, the utilization of heterologous herpesvirus for humans is considered untenable medically or ethically. Antigens Expressed in Live, Non-HSV Vectors Vaccinia virus has been proposed as a vector for delivering antigens to animals or humans (268). The principle of inserting foreign genes into a vaccinia vector has been exploited for the expression of the gD and gB genes of HSV (269–275). Significant concern has been raised over the utilization of vaccinia as a vector for delivering foreign antigens. In large part this concern stems from the occurrence of vaccinia gangrenosum and disseminated vaccinia in individuals who were vaccinated to prevent smallpox. As such, this major concern for adverse effects has led to decreased interest in utilizing vaccinia as a vector in the prevention of HSV, although this virus has become of increased importance with the threat of bioterrorism. Furthermore, immune memory in individuals who have previously received vaccinia may prevent recognition of any foreign gene insert. Adenoviruses have also been proposed as expression vectors, on the grounds that they might be safer than vaccinia (276). Animal models show good efficacy when adenovirus expressing gD are administered (277). Newer adenovirus platforms have been found in the HIV research field to stimulate strong CD8 responses to the inserted foreign gene (278) rendering this platform attractive for HSV. The varicella-zoster virus vaccine strain has been engineered to express HSV glycoproteins. Two of these constructs have been shown to afford animals protection against experimental HSV-2 genital herpes (279,280). Genetically Engineered Herpes Simplex Viruses Molecular biology makes it possible to modify, almost at will, the genome of large DNA viruses and construct genetically engineered attenuated live viruses (39). Utilizing the technology developed by Post and Roizman (281,282), recombinant HSVs were constructed as a prototypes of HSV vaccines (283). These vaccines were engineered with the objective that they

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should: be attenuated, whether for primary inoculation or potential reactivation of latent virus; protect against HSV-1 or HSV-2 infections; provide serologic markers of immunization distinct from wild-type infections; and serve as vectors to express immunogens of other human pathogens. The construction of these viruses was based on the use of an HSV-1 [HSV-1(F)] as a backbone. The genome was deleted in the domain of the viral thymidine kinase (TK) gene and in the junction region between the unique long and short sequences to excise some of the genetic loci responsible for neurovirulence and to create convenient sites and space within the genome for insertion of other genes. Last, an HSV-2 DNA fragment encoding the HSV-2 glycoproteins D, G, and I was inserted in place of the internal inverted repeat. The purpose of type 2 genes was to broaden the spectrum of the immune response and to create a chimeric pattern of antibody specificities as a serologic marker of vaccination. The resulting recombinant, designated as R7017, had no TK activity and, therefore, would be resistant to acyclovir. Therefore, another recombinant was created, designated R7020, by insertion of the TK gene next to the HSV-2 DNA fragment. Since this virus expresses TK, it is susceptible to antiviral chemotherapy with acyclovir. When analyzed by restriction enzyme digestion, the DNA of the recombinants shows typical patterns, which enable their unambiguous identification. When evaluated in rodent models, the two constructs appeared considerably attenuated in their pathogenicity and ability to establish latency and were capable of inducing protective immunity. The recombinants did not regain virulence, nor did they change DNA restriction enzyme cleavage patterns when subjected serial passages in the mouse brain (283). It is remarkable that the TK deleted virus R7017 behaved similarly to the TK expressing virus R7020, since the deletion of this gene was thought to attenuate the virus. These results were corroborated by studies in owl monkeys (A. trivirgatus) (284). While 100 PFU of wild-type viruses administered by peripheral routes were fatal to the monkeys, recombinants given by various routes in amounts at least 105fold greater were innocuous or produced mild infections, even in the presence of immunosuppression by total lymphoid irradiation (283). Unfortunately, human studies with this vaccine were disappointing. The maximum dose of vaccine administered was 105 PFU, which elicited only mild immunogenicity even with the administration of two doses (284). The ability to pursue higher doses of vaccine was limited because of an inability to produce satisfactory concentrations of vaccine. In many respects, the R7020 construct was overly attenuated. However, as noted below, this virus is now being studied for gene therapy of adenocarcinoma metastases from the colon to the liver. Regardless, these same principles of genetic engineering have been applied to newer the generation of newer constructs. The identification of a neurovirulence gene, identified as g134.5, provided a marker for genetic engineering (285). The deletion of the two copies of this gene and genes UL 55 and 56, genes associated with nuclear associated proteins, results in an attenuated candidate vaccine that is currently undergoing evaluation in animal models (158). These engineered viruses have been evaluated for gene therapy of malignancy, and therefore can also be assessed for the ability to induce host immune responses. An alternative strategy for attenuation pioneered by Minson and colleagues (286) involves deleting a gene essential

for HSV replication but providing the missing gene product in trans. Under these circumstances the vaccine virus is capable of infecting a cell and undergoing a single round of replication but it cannot produce replication-competent progeny because it lacks the essential gene. A gH deleted HSV-2 construct developed by Cantab Pharmaceuticals was shown to protect animals against experimental genital herpes and to have some therapeutic effect in a model of experiment recurrent genital herpes (287). Phase I studies showed the vaccine was safe and induced both cellular and humoral immune responses (288). The vaccine was evaluated in a randomized controlled trial for the treatment of recurrent genital herpes (289). The study involving 485 healthy volunteers who had six or more recurrences of genital herpes per year failed to show any effect on the frequency or duration of clinically apparent recurrences or on asymptomatic viral shedding for one year. While the vaccine was not effective for therapeutic use, its safety and immunogenicity profiles make it an excellent candidate for assessment as a prophylactic vaccine. Other replication impaired deletion mutants being developed by Knipe and colleagues show promise in animal model systems (290). Nucleic Acid–Based HSV Vaccines DNA vaccines induce immunity by a novel mechanism, the transfer of genetic material encoding an antigen to the cells of the vaccine recipient. Studies of experimental HSV DNA vaccines have shown them to be immunogenic and effective in animal models (291,292). While DNA vaccines have shown promising results in small animal models they have generally failed to induce protective immunity in primates and humans (the so-called primate barrier). However, recent studies utilizing DNA on gold beads administered through particlemediated epidermal delivery (293) have shown that low doses of DNA vaccines can induce protect levels of antibody in humans (294). On the basis of these findings Powdermed (now Pfizer Vaccines) has begun phase I testing of HSV DNA vaccines and other DNA vaccines are in preclinical development. Genetically Engineered Replication Attenuated HSV Genetically engineered HSV have mainly been assessed for the treatment of human glioblastoma multiforme. These constructs have included mutations in the viral genes thymidine kinase, DNA polymerase, ribonucleotide reductase, and g134.5 (295– 301). Each of these studies sought to optimize the therapeutic index in the treatment of gliomas by exploring therapies with different types of genetically engineered HSV constructs. While virtually any alteration of HSV ameliorates neurovirulence, only the deletions in the g134.5 gene consistently demonstrate safety and efficacy in animal models. Tumoricidal effects in vitro and in vivo in multiple glioma models (mouse, rat, and human glioma cell lines, human glioma explants) are demonstrable. In vivo models include tumor reduction in subrenal capsule and flank subcutaneous implants but, more importantly, increased survival and some tumor cures in intracranial implant models. These effects are reproducible in vivo for both immune deficient animals (nude, scid mice) (295,296,300,301) as well as immune competent models (rats and mice) (298,299,302,303). Animal Model Studies Studies in animal models of gliomas of various constructs of HSV (engineered viruses deleted in g134.5) have been performed. These studies demonstrate the following principles:

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(i) the time course of infection (quantitative virology and PCR) represents impaired replication with limited spread of virus across the brain using marker genes (lacZ under an ICP6 promoter) with HSV antibody staining (298,299,303,304), (ii) two selected mutations appear to avoid second-site mutations with reversion to wild-type phenotype (g134.5 and ribonucleotide reductase deletions) (305), (iii) the retention of the native HSV TK allows for acyclovir susceptibility (301), (iv) the safety of these constructs was established in susceptible primates (Aotus) (305), and (v) HSV could be used to successful vector genes (306–308), as reviewed (309). Indeed one construct, namely G207 (295) demonstrated an adequate safety profile in both cell culture as well as in animal models (310,311) and was efficacious in several tumor models in vivo (295–301). This candidate therapeutic is deleted in both copies of the g134.5 gene as well as ribonucleotide reductase. Sufficient quantities of virus were produced under GMP conditions a phase I study in humans with recurrent glioblastomas, as described below. Numerous other constructs have been developed, including cytokine/chemokine genes, enzymes, and receptors (312). However, other investigators have taken the approach of altering host recognition of HSV by deleting the a-47 gene and, thereby, allowing host MHC-I processing. The potential utility of this approach remains to be established. Clinical Experience with Intratumoral HSV in Glioma Patients The leading genetically engineered HSV candidates for treatment of glioblastomas is G207 and 1716, conditionally replicating HSV mutants. G207 was evaluated in a phase I safety trial for patients with recurrent malignant gliomas, failing standard therapy, and with a lesion greater than one centimeter in diameter (313). A total of 21 patients were recruited and received escalating doses of G207, beginning at 1  106 and in cohorts of three to a final dose of 3  109 PFU at five intratumoral sites. While adverse events were noted in several patients, no toxicity or adverse event was unequivocally ascribed to G207 administration. Importantly, no patient developed herpes simplex encephalitis. Host seroconversion to HSV was documented in one of five seronegative volunteers. Two volunteers have survived greater than three years with stable Kornofsky scores. These data provide the basis for a phase I B and II clinical trials, recently approved by the FDA, for further dose escalation after tumor debridement or administration of concomitant radiotherapy. Mutant 1716 is deleted in both copies of the g134.5 gene. Mutant 1716 has been studied in Scotland in a similar population of patients. In this trial, a total of nine patients were evaluated at one of three doses of virus, beginning at 1  103 and escalating by a factor of 10 to 1  105 (260). As in the United States study, there were no reports of significant adverse events directly attributable to virus administration. Four of the nine patients were alive 14 to 24 months after injection. Of note, the maximum amount of virus administered in this trial was four logs lower than that of the study performed in the United States. These promising studies have led to phase II trials in both the United States and the United Kingdom. Although the two trials utilized different genetically engineered constructs and doses of virus for administration, the demonstration of safety following intratumoral inoculation is truly remarkable and paves the way for the evaluation of genetically engineered HSV in phase II trials. Importantly,

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future studies should address the extent and magnitude of viral replication in the tumor as well as the host response in much more detail. Follow-up Investigations In studies in Europe and the United States, phase II investigations of genetically engineered HSV for the treatment of brain tumors are currently in progress. Second generation constructs that express the cytokine IL-12 will be in human investigations in the immediate future. This later construct will assuredly induce an enhanced effect within the tumor bed. Such studies will provide the groundwork for using a similar construct as a vaccine to prevent HSV infections.

CONCLUSION Within the last several years, focused efforts on developing vaccines for HSV infections as well as utilizing HSV constructs for cancer therapy have led to creative potential candidates. These vaccines have entered human investigations, which should indicate their potential efficacy. It has been learned, for example, that seronegative individuals at high risk for infection represent ideal candidates for participation in vaccine trials, while individuals with frequent recurrences probably do not offer the opportunity for complete suppression of symptomatic disease. As a consequence, vaccination should be scheduled for a time prior to exposure of the offending pathogen. For a vaccine designed to prevent HSV-2 infections, this would be early in adolescence prior to the onset of sexual activity. Adequate methodology has not always been applied to previously performed evaluations of HSV vaccines. The current and future studies are to be randomized, double blind, placebo controlled, with a sufficient number of volunteers for appropriate statistical analyses to comply with proper trial drugs. Interim analyses predicated on results obtained during the performance of the trial will guarantee the ethical nature of the trial design. After enrollment into a prospective clinical trial, the diversity of clinical HSV diseases, and the lack of predictability of patterns of recurrence, will mandate a very careful prospective evaluation for both symptomatic and asymptomatic evidence of infection in vaccine recipients, including the use of PCR to evaluate HSV shedding in vaccinees who become infected. On both clinical and laboratory levels, detailed evaluations will have to determine presence or absence of subsequent wild-type infection. Nevertheless, the intellectual and scientific challenges posed by the development of a vaccine to prevent HSV infections are extremely rewarding. It is hoped that within the next several years the results of excellent clinical trials will help establish the value or failure of vaccines to alter the natural history of HSV infections.

ACKNOWLEDGMENT Studies performed by the author and reported herein were initiated and supported under a contract (NO1-AI-30025, NO1AI-15113, NO1-AI-62554) with the Development and Applications Branch of the National Institute of Allergy and Infectious Diseases (NIAID), a Program Project Grant (PO1 AI 24009), by grants from the General Clinical Research Center Program (RR-032) and the State of Alabama.

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287. Boursnell ME, Entwisle C, Blakeley D, et al. A genetically inactivated herpes simplex virus type 2 (HSV-2) vaccine provides effective protection against primary and recurrent HSV-2 disease. J Infect Dis 1997; 175(1):16–25. 288. Roberts JSC, Uttridge JA, Hickling JK, et al. Safety, tolerability and viral containment of a disabled infectious single cycle HSV2 vaccine evaluated in phase I clinical trials in HSV-2 seropositive and seronegative volunteers. Abstracts of the 8th International Congress of Infectious Diseases, Boston, Massachusetts, 1998:89. 289. de Bruyn G, Vargas-Cortez M, Warren T, et al. A randomized controlled trial of a replication defective (gH deletion) herpes simplex virus vaccine for the treatment of recurrent genital herpes among immunocompromised subjects. Vaccine 2006; 24 (7):914–920. 290. Dudek T, Knipe D. Replication-defective viruses as vaccines and vaccine vectors. Virology 2006; 344(1):230–239. 291. Bourne N, Milligan G, Schleiss M, et al. DNA immunization confers protective immunity on mice challenged intravaginally with herpes simplex virus type 2. Vaccine 1996; 14(13):1230–1234. 292. Manickan E, Rouse R, Yu J, et al. Genetic immunization against herpes simplex virus. Protection is mediated by CD4þ T lymphocytes. J Immunol 1995; 155(1):259–265. 293. Fuller D, Loudon P, Schmaljohn C. Preclinical and clinical progress of particle-mediated DNA vaccines for infectious diseases. Methods 2006; 40(1):86–97. 294. Roberts L, Bark L, Fuller D, et al. Clinical safety and efficacy of a powdered hepatitis B nucleic acid vaccine delivered to the epidermis by a commercial prototype device. Vaccine 2005; 23 (40):4867–4878. 295. Martuza RL, Malick A, Markert JM, et al. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science 1991; 252(5007):854–856. 296. Markert JM, Malick A, Coen DM, et al. Reduction and elimination of encephalitis in an experimental glioma therapy model with attenuated herpes simplex mutants that retain susceptibility to acyclovir. Neurosurgery 1993; 32(4):597–603. 297. Boviatsis EJ, Scharf JM, Chase M, et al. Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes simplex virus vectors defective in thymidine kinase or ribonucleotide reductase. Gene Ther 1994; 1(5):323–331. 298. Jia WW, McDermott M, Goldie J, et al. Selective destruction of gliomas in immunocompetent rats by thymidine kinase-defective herpes simplex virus type 1. J Natl Cancer Inst 1994; 86(16): 1209–1215. 299. Kaplitt MG, Tjuvajev JG, Leib DA, et al. Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats. J Neurooncol 1994; 19(2):137–147. 300. Mineta T, Rabkin SD, Martuza RL. Treatment of malignant gliomas using ganciclovir-hypersensitive, ribonucleotide reductasedeficient herpes simplex viral mutant. Cancer Res 1994; 54(15): 3963–3966. 301. Chambers R, Gillespie GY, Soroceanu L, et al. Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma. Proc Natl Acad Sci U S A 1995; 92(5):1411–1415. 302. Mineta T, Markert JM, Takamiya Y, et al. CNS tumor therapy by attenuated herpes simplex viruses. Gene Ther 1994; 1(suppl 1): S78. 303. Boviatsis EJ, Park JS, Sena-Esteves M, et al. Long-term survival of rats harboring brain neoplasms treated with ganciclovir and a herpes simplex virus vector that retains an intact thymidine kinase gene. Cancer Res 1994; 54(22):5745–5751. 304. Boviatsis EJ, Chase M, Wei MX, et al. Gene transfer into experimental brain tumors mediated by adenovirus, herpes simplex virus, and retrovirus vectors. Hum Gene Ther 1994; 5 (2):183–191. 305. Yazaki T, Manz HJ, Rabkin SD, et al. Treatment of human malignant meningiomas by G207, a replication-competent

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62 Development of Vaccines to Prevent Group A Streptococcal Infections and Rheumatic Fever J. B. Dale University of Tennessee Health Science Center, Veterans Affairs Medical Center, Memphis, Tennessee, U.S.A.

M. Batzloff, Michael F. Good, and J. McCarthy Queensland Institute of Medical Research, Queensland, Australia

J. R. Carapetis Menzies School of Health Research, Charles Darwin University, Darwin, Australia

P. P. Cleary University of Minnesota, Minneapolis, Minnesota, U.S.A.

V. Fischetti and J. Zabriski Rockefeller University, New York, New York, U.S.A.

THE BURDEN OF GROUP A STREPTOCOCCAL DISEASES AND THE NEED FOR A VACCINE The world needs an effective, safe, and affordable vaccine to prevent group A streptococcal (GAS) infections and their sequelae for two reasons. First, the burden of disease is sufficient to make prevention a priority, and second, there is currently no effective strategy at a population level for primary prevention. GAS causes the broadest spectrum of disease of any bacterium. Infections range from the very common but relatively mild (pharyngitis or impetigo) to the less common but very severe (e.g., bacteremia, necrotizing fasciitis) that may be complicated by toxin-mediated illness (scarlet fever or streptococcal toxic shock syndrome). Following infection, a number of autoimmune sequelae may occur (rheumatic fever [RF] and poststreptococcal glomerulonephritis) and other parainfectious phenomena have also been described (poststreptococcal reactive arthritis, pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection). A systematic review of available data concluded that there are more than 18 million people with a chronic complication of a severe GAS disease worldwide, 15.6 million of whom have rheumatic heart disease (RHD). Another 1.8 million cases of severe disease occur each year. A total of 517,000 deaths annually are estimated to be due to this organism (1). This burden of severe disease is predominantly due to two clinical entities: rheumatic heart disease (>280,000 new cases and 230,000 deaths each year) and invasive infections (663,000 cases and 163,000 deaths each year). On top of this, there are more than 100 million new cases of impetigo and more than 600 million new cases of pharyngitis due to GAS each year. In absolute number, GAS infections and their complications primarily afflict the poor. Approximately 97% of GAS diseases

and associated deaths occur in developing countries (1). The association with poverty is due to overcrowded, unhygienic living conditions that promote bacterial transmission and poor access to medical care for treatment of infections and prevention of their complications (2). The highest prevalence rates of rheumatic heart disease are found in sub-Saharan Africa, Pacific nations, the Indian subcontinent, and indigenous populations in Australia and New Zealand (1). Data on invasive GAS infections are more limited, but the highest rates have been found in East Africa, Pacific nations, and indigenous people in Australia and the United States (3–6). Although developing countries bear the overwhelming burden of GAS diseases, this bacterium is also an important pathogen in affluent countries. Data from North America, Europe, and Australia show remarkably consistent all-age incidence rates of invasive GAS infections of 3 to 3.5 per 100,000, and mortality rates ranging from 7% to 15% (compared with *11/100,000 and 25%, respectively, in developing countries) (1). In the United States alone during 2005, these infections are estimated to have affected 10,700 people and caused 1500 deaths (7). GAS pharyngitis continues to be a common affliction of childhood, affecting approximately 12% of schoolaged children each year in the United States and Australia, (1,8). with dramatic economic consequences as a result of health care and days lost from school and work. These data confirm that GAS is an important cause of mortality and long-term morbidity. Indeed on the basis of data available on the World Health Organization website, GAS was the ninth most common single-pathogen cause of death in the world in 2002 (1). All the eight more common mortalityassociated pathogens have either vaccines available (tuberculosis, pneumococcus, hepatitis B, Haemophilus influenzae type b,

Chapter 62: Group A Streptococcal Infections and Rheumatic Fever

measles, rotavirus) or very intensive, well-funded vaccine development programs (HIV, malaria). An important feature in most developing countries and other populations with high rates of GAS infection is the enormous diversity of different strains present in a community at any one time compared with low-incidence populations, and the rapidity with which these strains enter and leave even small populations (9–11). Over periods of just months, the profile of strains, as defined by emm type or multilocus sequence type, can virtually entirely change (10,12,13), which presents a significant impediment to vaccine development.

ANTIBIOTIC PREVENTION OF GAS INFECTIONS AND THEIR SEQUELAE Globally, the only preventive measure that has been proven to be both effective and cost-effective at the population level for GAS diseases is secondary prophylaxis to prevent recurrent attacks of rheumatic fever (14). Indeed, rheumatic heart disease control programs are advised to focus on delivering secondary prophylaxis and good clinical care to people already known to have a history of rheumatic fever before implementing programs for primary prevention (15,16). Primary prophylaxis is the timely administration of antibiotics, usually penicillin, to people with GAS pharyngitis with the aim of preventing rheumatic fever. This strategy is highly effective at preventing cases of rheumatic fever that would otherwise have developed following a streptococcal sore throat (17). However, translating this knowledge into a practical strategy at the population level, particularly in developing countries, has proved to be almost impossible (18). The reasons for this are uncertain but may include a lack of resources, facilities, and trained staff to provide accurate diagnosis and treatment of sore throat at the primary care level, a tendency for people with sore throat not to seek health care in some communities, or a genuinely low incidence of streptococcal pharyngitis in particular populations (14,18,19). But even if all streptococcal sore throats could be diagnosed and treated, this would probably only prevent the minority of rheumatic fever cases, given that most rheumatic fever cases do not follow a significant sore throat (20). This has led some researchers to seek alternative approaches to primary prevention, including attempts to colonize the throats of young children with commensal streptococci in an attempt to prevent infection with GAS (21). It has also been hypothesized that, in some populations, streptococcal impetigo may have a causative role in rheumatic fever, directly or indirectly by either priming the immune response or acting as a reservoir of strains that cause disease by subsequently infecting the upper respiratory tract (22). Although primary prophylaxis may work to prevent rheumatic fever in individuals with a sore throat, there is no evidence that this approach will prevent pharyngitis-associated poststreptococcal glomerulonephritis or invasive disease. In developing countries, streptococcal skin infection underlies most cases of poststreptococcal glomerulonephritis and is also the major risk factor for invasive disease. Community-based programs to control skin infections may therefore help to reduce the rates of these serious sequelae, although this is unproven. Unlike primary prophylaxis of streptococcal pharyngitis to prevent rheumatic fever, there is no evidence that treatment of established skin infection will prevent sequelae that would otherwise have developed (14).

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Therefore, there is no systematic cost-effective approach that can currently be recommended for the primary prevention of most GAS diseases. Although research into alternative strategies is ongoing, no new methods are on the horizon in the short to medium term. It seems likely that the only effective large-scale strategy for the prevention of GAS infections will be a vaccine.

WHAT ARE THE FEATURES OF AN IDEAL GAS VACCINE? To serve the populations at highest risk of GAS diseases, a vaccine should either prevent serious diseases (rheumatic fever, invasive disease, and poststreptococcal glomerulonephritis) or as a minimum prevent infection at both of the major primary sites—upper respiratory tract and skin—that underlie these sequelae. It should offer immunity against the majority of circulating strains, and this immunity should withstand the rapid emergence of new strains. It should offer protection against disease that occurs in the first few years of life (e.g., invasive disease, impetigo) but also protection that lasts through the school-age years, when pharyngitis and rheumatic fever are most common. It should be safe, given the theoretical concerns that a GAS vaccine may have the potential to increase, rather than reduce, the risk of subsequent rheumatic fever (23). And the experiences with Hepatitis B, H. influenzae type b, and pneumococcal conjugate vaccines highlight the critical importance of developing a strategy to provide vaccine at an affordable price to developing countries right from the start.

STREPTOCOCCAL C5a PEPTIDASE: SEROTYPE-INDEPENDENT PROTECTION AGAINST GAS INFECTIONS Genotyping of clinical isolates has dramatically increased the number of serotypes and reaffirmed that dominant M serotypes vary significantly over time, in local populations, and among different regions of the world (19,24–26). Epidemiological studies reported at The XVIth Lancefield International Symposium on Streptococci and Streptococcal Diseases in Australia showed vast differences in serotypes responsible for more serious disease in various countries, and that higher numbered or newer serotypes can be common (25,26). Although no direct evidence exists in humans, it is assumed that serotype-specific immunity is the selective pressure that drives genetic variability in M proteins. Newer studies have discovered that N-terminal variable domains of M protein can have other functions (27). The past 15 years of molecular studies has uncovered, aside from variation in M protein, incredible genetic variability in the spectrum of other surface proteins expressed both between and within M serotypes. The relative plasticity of the GAS genome, and particularly emm genes, suggests that protective immunity may involve a moving target and indicates that a vaccine must either contain multiple type-specific M epitopes or a combination of proteins, some of which are conserved across all serotypes. The surface-bound streptococcal C5a peptidase (SCPA) is highly conserved across all emm genotypes tested and among other b-hemolytic streptococcal species associated with human disease (28–30). Since discovery of the C5a peptidase (28,31) experiments established that streptococci have the potential to destroy C5a chemotaxin, an early inflammatory signal at the bacterial surface and impede influx of phagocytes to initial foci of infection (32,33). Delayed phagocyte

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recruitment is believed to provide GAS a window of time to translocate into lymphoid tissue where bacteria then replicate and expand their numbers. Both subdermal and intranasal murine models of infection compared mutant strains devoid of SCPA or SCPB (associated with group B streptococci) and confirmed this concept (33–35). A surprising observation was that M protein had little or no measurable effect on clearance of streptococci from the oral-nasal mucosa relative to SCPA (33). Consistent with that surprise are recent microarray studies of genes activated by intranasal infection. These experiments revealed that streptococci devoid of SCPA induce significantly fewer genes associated with the innate immune response than wild-type streptococci or mutants defective in M1 protein expression (Cleary, unpublished data). Early experiments showed that intranasal immunization with affinity-purified recombinant SCPA protein derived from M49 streptococci (36) and administered without adjuvants induced strong salivary and serum immunoglobulin (Ig)A and IgG responses in mice. Throat cultures following intranasal challenge demonstrated that immunized animals cleared streptococci more rapidly than nonimmunized controls. Cross-protection against serotypes M2, M11, M1, and M6 was also demonstrated (36). A key question to be addressed was whether parenterally administered antigen would protect against an intranasal challenge of streptococci. The SCPA1 gene from an M1 streptococcal strain was cloned, genetically inactivated, and the mutant protein (SCPAw) was expressed in Escherichia coli (37). Protection studies again made use of the intranasal infection model in which mice were vaccinated by subcutaneous (SC) injection with SCPAw protein, mixed with Alum and MPL adjuvants. Mice immunized with either recombinant SCPAw or SCPA49 cleared intranasally administered serotype M1 or M49 streptococci more efficiently than control mice that had been immunized with tetanus toxoid. Moreover, mice immunized with SCPAw vaccine protein were also protected from pneumonia following introduction of M1 streptococci into lungs (38). It was assumed that protection was dependent on antibody that neutralized C5ase enzymatic activity; experiments confirmed that sera from mice or rabbits immunized with SCPAw protein blocked the enzymatic activity of SCPA. Recent analyses of human sera demonstrated that the neutralization potential correlates with the concentration of SCPA-specific IgG (Cleary, unpublished). The primary reservoir for GAS is most likely school children. Therefore, to be fully effective and induce herd immunity, a vaccine must prevent colonization in uninfected children before this population becomes infected and serves as a source for dissemination of bacteria to other individuals. However, it is unclear whether antibody to any streptococcal antigen has the potential to eliminate streptococci from tonsils of carriers. Nasalassociated lymphoid tissue (NALT) of mice is functionally similar to human palatine tonsils and very susceptible to infection following intranasal inoculation (34). An intranasal NALT infection model was adapted to investigate whether immunization with recombinant SCPA influenced streptococcal clearance or colonization of this secondary lymphoid tissue and whether antibody to SCPA can prevent infection of NALT. For active immunization experiments, SCPAw or tetanus toxoid proteins were administered intranasally, mixed with cholera toxin, a known enhancer of secretory antibody responses (39). In these experiments persistence of streptococci was assessed either by measuring light emanating from the nose (when infected with a luciferase positive M49 strain) or by viable counts of streptococci specifically associated with dissected NALT tissue. As predicted,

Figure 1 Anti-SCPA antibody promoted clearance of Luxþ M49 streptococci by mice. Sera were administrated intranasally to mice two hours before challenge with 7  107 CFU of GAS. Streptococci were also preincubated with sera before intranasal inoculation of the mice. Sera were pooled from mice immunized with recombinant SCPAw protein with and without cholera toxin or with PBS. Abbreviations: SCPA, surface-bound streptococcal C5a peptidase; GAS, group A streptococci; PBS, phosphate-buffered saline.

intranasal vaccination of mice with SCPAw protein prevented infection of NALT tissue by GAS. Intranasal application of mouse or rabbit sera that contained high titers of anti-SCPA antibodies also prevented NALT infection following intranasal challenge (Fig. 1) (39). Although the above results suggest that specific antibodies induced by vaccination with SCPAw protein are responsible for immunological protection, there is no proof that neutralization of C5ase activity is the mechanism of protection. SCPA protein has other activities that could potentially contribute to streptococcal virulence. It binds to human epithelial cells and fibronectin with high affinity and it promotes invasion of epithelial cells by group B Streptococcus (35). Rabbit anti-SCPA or anti-SCPB sera inhibit in vitro ingestion of GAS (Cleary, unpublished) and group B streptococci (40), respectively, by A549 epithelial cells. These findings raise the possibility that antibody prevents translocation of streptococci across the nasal mucosa into NALT, a process which depends in part on uptake of streptococci by mucosal M cells. Blockage of streptococcal uptake by these cells could be important for protection. Vaccines based on SCPA or conserved regions of M protein have the potential to prevent infections by other species of b-hemolytic streptococci. Substantial epidemiological data indicate that C and G streptococci are also associated with pharyngitis, and more rarely sepsis and other serious complications (29,40,41). Studies from India found that group G streptococci are isolated more often from patients with pharyngitis than are GAS (25,26). Moreover, human isolates of group C and G streptococci produce many of the same virulence factors as GAS, including M protein and superantigens. Human isolates of C and G streptococci also uniformly produce SCPA-like proteins (30,40,41). Hill and colleagues were the first to demonstrate production of a C5ase enzyme by group B streptococci (31), and subsequent studies demonstrated that all serotypes have the potential to produce SCPB proteins, which are 95% identical in sequence to those produced by GAS (29,42). A vaccine that also reduced the incidence of infection by these species would be especially attractive. Anti-SCPB antibody is opsonic for group B streptococci in whole blood, and induces killing of these streptococci by primary bone marrow macrophages (38,43). SCPB was

Chapter 62: Group A Streptococcal Infections and Rheumatic Fever

also found to be an exceptional carrier protein when conjugated to serotype III capsular polysaccharide. Without a protein carrier, group B streptococcal capsular polysaccharides are virtually nonimmunogenic. SC vaccination of mice with SCPB conjugates induced serotype-independent protection against lung infection by both GAS and group B streptococci. Sera from these mice promoted serotype-independent opsonization and macrophage killing of group B streptococci (38,43). Safety issues have historically influenced GAS vaccine development and continue to hold center stage. A primary reason is that the biological basis for rheumatic fever and other autoimmune reactions associated with GAS infection is not fully understood. It has been argued that induction of autoimmune responses by C5a peptidase proteins is unlikely because autoimmune disease in humans or animals has not been associated with group B streptococcal infections. This epidemiological observation strongly suggests that neither SCPB nor SCPA proteins will induce a pathological, tissue cross-reactive immune response. Moreover, SCPA protein did not induce tissue cross-reactive antibodies in rabbits or mice following vaccination with purified recombinant protein. The incidence of GAS pharyngitis is generally lower in older children and adults, but the immunological mechanism for increased resistance to infection in these age groups is unknown (44). In the past, trials of other vaccines were often begun without clear immune correlates with protection. Streptococcal vaccines today face a similar challenge. To date, no correlation between an immune response to any specific streptococcal antigen and protection in human subjects exists. The common failure of children to develop a protective immune response following pharyngitis or impetigo is another poorly understood concern. In fact, a third of children studied failed to have an increase in anti-streptolysin O antibodies or other streptococcal antigens that have been evaluated (45). Adults are thought to be generally more immune to infection; however, resistance clearly either decreases with age or fluctuates over time. In a small study, more than 70% of saliva from adults contained measurable levels of SCPA-specific secretory Ig (sIg)A and IgG antibodies (46). This contrasted sharply with children under 10 years old, where less than 15% of salivary samples contained SCPAspecific antibody. Measurements of SCPA-specific antibody in acute and convalescent sera also confirmed that children mount an antibody response to SCPA following infection, yet a prospective study to correlate antibody levels with resistance to subsequent infection has not been done (45). Evidence in murine models supports inclusion of SCPA protein in a vaccine to prevent streptococcal pharyngitis: (i) Intranasal immunization induces IgG and IgA responses and reduces colonization by streptococci following intranasal infection, (ii) intranasal administration of high titer antibody prevents colonization of mouse NALT, (iii) SC injections of recombinant SCPA with adjuvants induces a strong IgG response that speeds clearance of streptococci from the oralnasal mucosa, and (iv) either intranasal or parental routes of administration of this single protein induce protection against multiple serotypes of group A and group B streptococci.

GAS CARBOHYDRATE AS A VACCINE CANDIDATE The general dogma for over 60 years has been that the broadbased immunity to GAS infections (44) that appears with increasing age is due to the presence of multiple type-specific

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antibodies in the serum of these individuals. Yet, as evidenced by Dr. Lancefield’s studies, the presence of multiple type-specific M protein antibodies in human sera is actually quite rare (47). In view of these observations, investigators searched for other antigens that might induce broad-based immunity and antibodies prepared against these antigens that might be both phagocytic and protective in an animal challenge model. One report (48) indicated that antibodies to the group A carbohydrate (GRA) CHO increased with age and were phagocytic for multiple serotypes of GAS, and specific removal of the antibody (leaving all others present in the serum) resulted in loss of opsonic activity. Furthermore, elution of affinity-purified antibodies from N-acetylglucosamine-coated beads restored the majority of opsonic activity of the original antiserum. Several new questions have now been addressed: (i) will GRA CHO antibodies passively protect in a mouse challenge model, (ii) will active immunization with the GRA CHO protect against streptococcal challenge infections, (iii) do GRA CHO antibodies increase with age and correlate with the presence or absence of GAS in the throats of normal school children, and (iv) is there any evidence that GRA CHO induces antibodies that cross-react with human tissues? (i) Table 1 demonstrates that rabbit sera obtained from animals immunized with GRA CHO conjugated to tetanus toxoid (TT) provided passive protection following lethal GAS challenge infections in mice. (ii) Animals were actively immunized subcutaneously with an average of four doses of streptococcal GRA CHO conjugated to TT. Three different Mþ strains were used in these experiments and the number of organisms needed to kill approximately 100% of the control animals is shown in Table 2. Immunization with GRA CHO significantly decreased the number of deaths in immunized mice when compared with controls. (iii) A study of 300 normal Mexican children has shown that the anti-CHO antibody titers increase with age and these titers correlate with the presence or absence of GAS colonization in the throat. The titers of other antistreptococcal antibodies were similar in both groups. In this study of children 5 to 14 years of age, it was determined that approximately 20% carried GAS in their throats. Anti-CHO antibodies were measured in those children with positive GAS cultures and compared with those who had negative cultures. Table 1 Passive Protection Test in Balb/c Mice Against Group A Streptococcus Type 6 (S43/46) Serum

Colonies

Micea,b

NRS Group A carbohydrate Ab

200–500 200–500

3/26bb,c 16/26

a

Number of mice survived/injected. Similar protection was seen against type 3 (D58/93/7). p < 0.001. Source: From Ref. 49. b c

Table 2 Active Immunization Studies with Group A Streptococcal Carbohydrate in Mice Challenged with Live Type 14 (S23) Streptococcia Group

Adjuvant

Inoculum range

Survived/injected

Carbohydrate-TT conjugate TT

Alum

3–3.6  105

18/23b

Alum

3–3.6  106

5/22

a

þ

Similar protection was seen against two other M types. p < 0.001. Source: From Ref. 49.

b

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Figure 2 (A) Anti-carbohydrate antibody titers in serum of children with positive and negative throat cultures from group A streptococci. Note the titers in the negative sera were twice as high as those with positive cultures. (B) Serum ASO and DNAse B titers in the same population. Note there was no significant difference in the titers of the antibodies in the sera of the patients whose throat cultures were positive or negative for group A streptococci. Source: From Refs. 37, 49. Abbreviation: ASO, anti-streptolysin O.

Figure 2A shows that the titers were twice as high in children with negative cultures when compared with children with positive cultures ( p < 0.003). Figure 2B demonstrates that other antistreptococcal antibodies were equally divided between the two groups, suggesting that the anti-CHO titers were uniquely increased in those children with negative throat cultures. Finally, the question of whether the GRA CHO antibodies cross-reacted with human tissue was addressed. These questions were originally raised by Dudding and Ayoub (50) and expanded on by Cunningham et al. (51). Using human frozen tissue from organs known to be involved in cross-reactions between streptococcal antigens and human tissues, no evidence was found that anti-CHO antibodies were directed to these human antigens measured both by immunofluorescence and by enzyme-linked immunosorbent assays (ELISAs) using known human cytoskeletal antigens (data not shown). In conclusion, the GAS CHO can elicit antibodies that are both phagocytic and protective against several different typespecific Mþ streptococcal strains. High titers of CHO antibodies are associated with decreased colonization of the throat by GAS, and the CHO antigen does not induce cross-reactive antibodies against human tissues. For these reasons a case can be put that GAS polysaccharide is an excellent candidate immunogen for the prevention of GAS infections.

M PROTEIN STRUCTURE AND FUNCTION Streptococcal M protein was identified over 70 years ago by Rebecca Lancefield (52). A review by Lancefield in 1962 (53) clearly described the studies carried out for nearly 35 years, defining this molecule as a major virulence factor for the Streptococcus because of its antiphagocytic property. In 1974, a review by Fox (54) delineated studies since Lancefield’s review and underscored the knowledge to that time of the structure, function, and immunochemistry of the M molecule. The streptococcal M protein is one of the best-defined molecules among the known bacterial virulence determinants. Clearly protective immunity to GAS infection is achieved through antibodies directed to the M protein (55,56), a major virulence factor present on the surface of all clinical isolates. M protein is a coiled-coil fibrillar protein composed of three major segments of tandem repeat sequences that extends nearly 60 nm from the surface of the streptococcal cell wall (55) (Fig. 3).

Figure 3 Proposed model of the M protein from M6 strain D471. The coiled-coil rod region extends about 60 nm from cell wall with a short nonhelical domain at the NH2-terminus. The Pro\Gly-rich region of the molecule is found within the peptidoglycan. The membrane-spanning segment is composed predominantly of hydrophobic amino acids and a short charged tail extends into the cytoplasm. Data suggests that the membrane anchor may be cleaved shortly after synthesis. The A-, B-, and C-repeat regions are indicated along with those segments containing conserved, variable, and hypervariable epitopes among heterologous M serotypes. Pepsin designates the position of a pepsin susceptible site near the center of the molecule. Source: From Ref. 57.

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The A- and B-repeats located within the N-terminal half are antigenically variable among the more than 120 known streptococcal types with the N-terminal nonrepetitive region and A-repeats exhibiting hypervariability. The more C-terminal C-repeats, the majority of which are surface exposed, contain epitopes that are highly conserved among the identified M proteins (58). Because of its antigenically variable N-terminal region, the M protein provides the basis for the Lancefield serological typing scheme for GAS (55). The M protein is considered the major virulence determinant because of its ability to prevent phagocytosis when present on the streptococcal surface, and thus, by this definition, all clinical isolates express M protein. This function may in part be attributed to the specific binding of complement factor H to both the conserved C-repeat domain (59) and the fibrinogen bound to the B-repeats (60), preventing the deposition of C3b on the streptococcal surface. It is proposed that when the Streptococcus contacts serum, the factor H bound to the M molecule inhibits or reverses the formation of C3b,Bb complexes and helps to convert C3b to its inactive form (iC3b) on the bacterial surface, preventing C3b-dependent phagocytosis. Studies have shown that antibodies directed to the B- and C-repeat regions of the M protein are unable to promote phagocytosis (61). This may be the result of the ability of factor H to also control the binding of C3b to the Fc receptors on these antibodies, resulting in inefficient phagocytosis (62). Antibodies directed to the hypervariable N-terminal region are opsonic, perhaps because they cannot be controlled by the factor H bound to the C-repeat region. Thus, it appears that the Streptococcus has devised a method to protect its conserved region from being used against itself by binding factor H to regulate the potentially opsonic antibodies that bind to these regions. When we found this, we reasoned that it could explain why even though adults are more resistant than children to streptococcal pharyngitis, they do get sporadic infections. If antibodies produced to the conserved region were in fact protective, then it would be expected that these antibodies would be protective to all serotypes encountered, since opsonic IgG is usually sterilizing (as is type-specific IgG). Since this was not the case, we suspected that another mechanism resulted in broad protection that could be occasionally breached.

DEFINING A MINIMAL CONSERVED EPITOPE IN THE C-REPEAT REGION OF THE M PROTEIN The pathogenesis of RHD is believed to involve an autoimmune process. In studies of acute valvulitis lesions, T cells have been identified (63); others have shown that such T cells can be stimulated by M protein peptides (64,65). It has also been shown that human T-cell clones derived from the peripheral blood of individuals with or without a history of RF or signs of RHD and grown in response to a peptide from the conserved region of the M protein are able to react to myosin or an extract of human heart tissue (66). These data suggest that while M protein–specific T cells may contribute to the pathogenesis of RHD, other factors are also required. The conserved region of the M protein can induce protection in animal models of GAS infection (67,68) and antibodies to conserved region peptides show opsonic properties when a method based on a modification of Lancefield’s original description is used (69). Thus, one approach to vaccine development has been to define minimal opsonic determinants on the conserved region of the M protein that do not stimulate

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potentially autoreactive T cells. Because the M protein is conformational, it is critical that minimal determinants be correctly folded to mimic native structure. To preserve the a-helical structure of the determinant, a process was developed to display minimal M protein sequences within flanking nonGAS sequences derived from another protein with known a-helical structure, the GCN4 DNA binding protein of yeast (70). Such peptides are referred to as ‘‘chimeric’’ peptides; two such peptides, ‘‘J8’’ and ‘‘J14,’’ were capable of inducing opsonic antibodies in mice without stimulating a T-cell response to the conserved region (71,72). The conserved epitope within the last repeat of the C-repeat region of the M protein, from which the chimeric peptides J8 and J14 are derived, is highly conserved across GAS strains and emm types. This region of the M protein (emm gene) has been subject to extensive DNA sequence analysis in a large number of clinical GAS isolates collected from regions with both high and low endemnicity of GAS, including Australia, India, Thailand, and Fiji. To date, only two variants, which differ by only three amino acids, have been identified. Recent data indicate that J8- and J14specific antisera raised in mice can recognize and bind either variant (unpublished). Mice vaccinated with the J8 and J14 chimeric peptides had enhanced survival compared with control cohorts following lethal systemic GAS challenge with homologous strains that express the identical conserved region epitope (73). In addition, mice vaccinated with the chimeric peptides also had enhanced survival compared with control cohorts following lethal GAS challenge with heterologous GAS strains that express the variant epitope (unpublished). Further work with these chimeric peptides has recently included the synthesis of chimeric peptides with lipid attachments [lipid-core peptide (LCP) or Pam2Cys] to form J8 and J14 containing lipopeptides (74,75), as represented in Figure 4A. These lipopeptides, when administered mucosally, can induce peptide-specific mucosal IgA and systemic IgG in mice. Lipopeptide vaccinated mice had significantly enhanced survival compared with control cohorts following systemic (75) and mucosal (74) GAS challenge. Mice vaccinated with these lipopeptides developed peptide-specific mucosal IgG and had reduced GAS colonization of the throat following intranasal challenge (see section below for more information on mucosal protection).

COMBINING CONSERVED AND SEROTYPIC DETERMINANTS OF M PROTEIN While a minimal conserved epitope can induce protection in mice against GAS challenge (71) and can induce antibodies capable of opsonizing multiple strains of GAS (78), it remains to be seen whether it will protect humans from GAS and RF/ RHD. Of importance is the paucity of detailed knowledge of how relevant this mouse model is to human infection. While a theoretical concern exists that antibodies specific for the minimal determinant may be poorly opsonic compared with antibodies to the serotypic determinants (71,78,79), human antibodies specific for the conserved region purified from the sera of GAS-exposed volunteers can opsonize GAS. Further, there is a strong association between age-related acquisition of resistance to GAS infection and age-related acquisition of serum antibodies to the conserved region of individuals living in communities highly endemic for GAS infection (80). Nevertheless, short synthetic peptides may induce in humans antibodies of a different fine specificity and with less antibacterial efficacy.

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Figure 4 Diagrammatic representation of (A) lipid-core peptide lipopeptide containing four copies of the chimeric peptide ‘‘J8’’ and (B) space-filling model for ‘‘heteropolymer’’ immunogen consisting of alkane backbone and pendant side chains representing M protein epitopes. Source: From Refs. 71, 76, and 77.

It could be argued that by combining a conserved epitope with a limited number of serotypic determinants representative of common strains, a more effective vaccine might result. The benefits derived from adding serotypic determinants would need to be balanced by the drawback that strains that are common still vary between localities. Many of the strains from Thailand, for example, are serologically ‘‘nontypeable’’ using reagents that can distinguish between strains common elsewhere. Similarly, many strains that are common among the Australian Aboriginal population are nontypeable using serotypic reagents and have required ‘‘emm typing’’ using a PCR-based approach (81). The N-terminal peptides of seven of these common Aboriginal isolates were combined with the J14 minimal epitope using novel polymer chemistry (77). The immunogen consisted of an alkane backbone and pendant side chains formed by the different epitopes. It is illustrated diagrammatically in Figure 4B.

Outbred mice were vaccinated with the polymer, and the sera of the mice contained antibodies specific for each of the individual epitopes. The sera opsonized two different strains of GAS, one of which had an N-terminal sequence in common with the vaccine and one that had an N-terminal sequence not present in the vaccine. Both strains had a conserved sequence identical to the epitope found on J14. These data suggested that both the N-terminal and C-terminal-specific antibodies contributed to the bactericidal activity of the serum. Following challenge with a lethal dose of either strain, the vaccinated mice were completely protected (Fig. 5). More recently heteropolymerinduced protection has been demonstrated using this mouse challenge model and clinical isolates of GAS containing a larger number of N-terminal sequences including strains from the Northern Territory of Australia that express either the variant or the conserved region epitope (unpublished).

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Figure 5 Heteropolymer with N-terminal and conserved peptide pendant side chains induces immunity to GABHS (group A b-hemolytic streptococci) challenge. The 88/30 (i) bacterial challenge strain is a clinical isolate from the Northern Territory of Australia and is represented on the heteropolymer construct by both an amino-terminal peptide and the conserved region epitope. In contrast, 2040 (ii) is a reference challenge strain that is only represented on the heteropolymer by the conserved region epitope. Source: From Ref. 71.

While these data are promising, a potential drawback of this approach is that while the polymerization technology utilizes consistent molar ratios of peptide, it cannot enable the specific ordering of the epitopes on the polymer backbone to be defined. Thus, it is expected that there would be batch-to-batch variation in the composition of the product, a factor that could affect immunogenicity and impede regulatory approval. New polymer chemistries are being developed that will enable the production of a product with a defined order of epitopes on the polymer. Alternative approaches would be to produce a polymer as a recombinant protein or as a DNA vaccine with all epitopes joined head to tail (see above).

MUCOSAL VACCINE FOR SEROTYPEINDEPENDENT PROTECTION School-age children are much more susceptible to GAS pharyngitis than adults. Furthermore, the siblings of a child with a streptococcal pharyngitis are five times more likely to acquire the organism than one of the parents. This decreased occurrence of streptococcal pharyngitis in adults might be explained by a nonspecific age-related host factor resulting in a decreased susceptibility to streptococci. Alternatively, protective antibodies directed to antigens common to a large number of GAS serotypes might arise as a consequence of multiple infections or exposures during childhood. This could result in an elevated response to conserved M protein epitopes. This latter hypothesis is partly supported by earlier studies on the immune response to the M protein where it was found that the B-repeat domain (Fig. 3) was clearly immunodominant (82). When rabbits were immunized with the whole M protein molecule, the first detectible antibodies were directed to the B-repeat region and rose steadily with time. It was only after repeated M protein immunization that antibodies were produced against the hypervariable A- and conserved C-repeat regions. Unlike antibodies to the N-terminal region, it was clear that antibodies directed to the exposed C-repeat region were not opsonic (61). Because of this, experiments were performed to explore whether mucosal antibodies to this conserved region of M protein could play a role in protection from infection.

Taking advantage of the pepsin cleavage site in the center of the M molecule (separating the variable and conserved regions) (Fig. 3), the recombinant M6 protein was cleaved and the Nand C-terminal fragments separated by SDS-PAGE and Western blotted. When the blots were reacted with different adult human sera, all adults tested had antibodies to the C-terminal conserved region while, as expected, only sera that were opsonic for the M6 organisms reacted with the N-terminal variable region (67,83,84). Similar studies performed with M proteins isolated from five different common serotypes (M3, M5, M6, M24, M29) revealed that sera from 10 of 17 adults tested did not have N-terminal-specific antibodies to these M types, while only two sera reacted with two serotypes and the remaining five sera with only one serotype. However, all sera tested reacted to the C-terminal fragment of the M molecule. Similar results were seen when salivary IgA from adults and children were tested in ELISA against the N- and C-terminal halves of the M6 molecule (V. A. Fischetti, unpublished data). In all, this is further evidence that the relative resistance of adults to streptococcal pharyngitis is clearly not due to the presence of type-specific antibodies to multiple types, but may perhaps be due to the presence of antibodies to conserved determinants. From these findings it was reasoned that an immune response to the conserved region of the M molecule might afford protection by inducing a mucosal response to prevent streptococcal colonization and ultimate infection. In view of the evidence that the conserved C-repeat epitopes of the M molecule are immunologically exposed on the streptococcal surface (58), it should be possible to generate mucosal antibodies that are reactive to the majority of streptococcal types using only a few distinct conserved region antigens for immunization.

PASSIVE PROTECTION AT THE MUCOSAL SURFACE sIgA is able to protect mucosal surfaces from infection by pathogenic microorganisms (85) despite the fact that its effector functions differ from those of serum-derived Igs (86). When streptococci are administered intranasally to mice, they are able

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Figure 6 The extent of colonization of mice challenged with group A streptococci after oral immunization with M protein–conserved region M6 peptides linked to CTB or CTB alone. Orally immunized mice were swabbed each day after challenge with M14 streptococci and plated on blood plates to determine the extent of colonization compared with mice vaccinated with CTB only. Plates showing group A streptococci were scored as positive. Abbreviation: CTB, cholera toxin B.

to cause death by first colonizing and then invading the mucosal barrier resulting in dissemination of the organism to systemic sites. Using this model, it was first examined if sIgA delivered directly to the mucosa plays a role in protecting against streptococcal infection. Live streptococci were mixed with affinity-purified M protein–specific sIgA or IgG antibodies and administered intranasally to the animals (87). The results clearly showed that the anti–M protein sIgA protected the mice against streptococcal infection and death, whereas the opsonic serum IgG administered by the same route was without effect. This indicated that sIgA can protect at the level of the mucosa and may preclude the need for opsonic IgG in preventing streptococcal infection. These studies were also one of the first to compare purified, antigen-specific sIgA and serum IgG for passive protection at a mucosal site. Passive protection against streptococcal pharyngeal colonization was also shown by the oral administration of purified lipoteichoic acid (LTA) but not deacylated LTA prior to oral challenge in mice (88). The addition of anti-LTA by the same route also protected mice from oral streptococcal challenge. While several in vitro studies showed the importance of M protein (89–91) and LTA (92) in streptococcal adherence, these in vivo studies, together with those presented above, suggest that both M protein and LTA may play a key role in the colonization of the mouse pharyngeal mucosa. However, it is uncertain whether this is also true in humans.

ACTIVE IMMUNIZATION AT THE MUCOSAL LEVEL WITH CONSERVED REGION PEPTIDES To determine whether a local mucosal response directed to the conserved exposed epitopes of M protein can influence the course of mucosal colonization by GAS, peptides corresponding to these regions were used as immunogens in a mouse model (83,84). Overlapping synthetic peptides of the conserved region of the M6 protein were covalently linked to the mucosal adjuvant cholera toxin B (CTB) subunit and administered intranasally to mice in three weekly doses and boosted 30 days after the last dose with the peptide mixture. Ten days later, animals were challenged intranasally with live streptococci (either homologous M6 or heterologous M14), and pha-

ryngeal colonization by the challenge organism was monitored for 10 to 15 days. Mice immunized with the peptide-CTB complex showed a significant reduction in colonization with either the M6 or M14 streptococci compared with mice receiving CTB alone (83,84) (Fig. 6). Thus, despite the fact that conserved region peptides were unable to evoke an opsonic antibody response (61), these peptides have the capacity to induce a local immune response capable of influencing the colonization of GAS at the nasopharyngeal mucosa in this model system. These findings were the first to demonstrate protection against a heterologous serotype of GAS with a vaccine consisting of the widely shared C-repeat region of the M6 protein. Confirmation of these findings was later published independently using different streptococcal serotypes as the immunizing and challenge strains (93). In a separate study, Pruksakorn et al., found (66), using a different criteria for streptococcal opsonization than previously published (47), reported that when a peptide derived from the conserved region of the M protein was used to immunize mice, it induced antibodies capable of opsonizing type 5 streptococci and streptococci isolated from Aboriginal and Thai rheumatic fever patients. These findings are in sharp contrast to the studies of Jones et al. (61), who showed that antibodies to the conserved region of M protein are not opsonic. However, since the peptide reported by Pruksakorn et al. (66) is similar to one of the peptides used by Bessen and Fischetti (83,84) in mucosal protection studies (see above), the induction of serum IgG during mucosal immunization may offer added protection against streptococcal infection.

VECTORING THE M PROTEIN WITH VACCINIA VIRUS To further verify the validity of using the M protein–conserved region as a streptococcal vaccine, experiments were repeated in a vaccinia virus vector system. In these studies, the gene encoding the complete conserved region of the M6 molecule (from the pepsin site to the C-terminus, see Fig. 3) was cloned and expressed in vaccinia virus producing the recombinant VV::M6 virus (94,95). Tissue culture cells infected with this

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Figure 7 The extent of colonization of mice challenged with group A streptococci after oral immunization with recombinant vaccinia virus containing the gene for the whole conserved region of the M6 protein or vaccinia virus alone. Orally immunized mice were swabbed each day after challenge with M14 streptococci and plated on blood plates to determine the extent of colonization compared with mice vaccinated with wild-type vaccinia only. Plates showing group A streptococci were scored as positive.

virus were found to produce the conserved region of the M6 molecule. Animals immunized intranasally with only a single dose of recombinant virus were significantly protected from heterologous streptococcal challenge compared with animals immunized with wild-type virus (Fig. 7). When the extent of colonization was examined in those animals immunized with wild-type or the VV::M6 recombinant, the VV::M6-immunized animals that exhibited positive swabs showed a marked reduction in overall colonization compared with controls, indicating that mucosal immunization reduced the bacterial load on the mucosa in these animals. Animals immunized intradermally with the VV::M6 virus and challenged intranasally showed no protection. The approaches described above proved that induction of a local immune response was critical for protection against streptococcal colonization and that the protection was not dependent on an opsonic response. However, in the event that the Streptococcus was successful in penetrating the mucosa and establishing an infection, only then would type-specific antibodies be necessary to eradicate the organism. This idea may perhaps explain why adults sporadically develop a streptococcal pharyngitis, that is, a mucosal response may be breached when a large number of streptococci are encountered on the mucosal surface. The success of these strategies not only forms the basis of a broadly protective vaccine for the prevention of streptococcal pharyngitis but may offer insights for the development of other vaccines. For instance, a vaccine candidate that does not evoke protective immunity by the parenteral route may prove to be successful by simply changing the site of immunization. Furthermore, these results emphasize the fact that in some instances antigens need to be presented to the immune system in a specific fashion to ultimately induce a protective response.

synthesized and exported at the septum, where new cell wall is also being produced and translocated to the surface (55,98). Thus, the C-terminal hydrophobic domain and charged tail in these proteins function to control the export and anchoring process by acting as a temporary stop to position the LPXTG motif [the anchor motif common to >100 surface proteins on gram-positive bacteria (Fig. 8) (99)] precisely at the outer surface of the cytoplasmic membrane. This sequence motif, which is an enzyme recognition sequence, is cleaved resulting in the attachment of the surface-exposed segment of the protein to a cellular substrate (96). This idea is supported by studies indicating that the C-terminal hydrophobic domain and charged tail are missing from the streptococcal surface M protein extracted from the cell wall (57,96). Since the anchor region is highly conserved among a wide variety of surface molecules within several different gram-positive species, could it be fused to a foreign antigen and used to deliver the resulting fusion protein to the surface of a gram-positive bacterium, ultimately anchoring it to the cell? To answer this, the streptococcal M protein was employed in a model system. Pozzi et al. (100,101) were the first to deliver a fusion protein to the surface of the gram-positive human oral commensal Streptococcus gordonii. The approach utilized knowledge

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Figure 8 Conservation of the LPXTG motif at the C-terminal end of surface proteins on gram-positive bacteria. Sixty-eight surface proteins from gram-positive organisms were compared as to the number of times L, P, T, and G were found. As seen, L and P are found 100% of the time in this position and T and G more than 86% of the time.

The importance of the C-terminal region in the attachment of surface proteins in gram-positive bacteria was previously demonstrated using the protein A from Staphylococcus aureus as a model system (96,97). Surface proteins in gram-positive bacteria (which could number more than 20 in a single organism) are

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Figure 10 Engineering a fusion protein for surface expression. On a plasmid, the surface-exposed region of the M protein is excised and replaced by a multiple cloning site. The foreign protein to be expressed on the bacterial surface is then inserted in frame in the multiple cloning site. The signal sequence (S) and a short segment of the M protein N-terminal sequence (N) are included in the construct to allow proper processing of the signal peptidase. The ermC antibiotic marker is used for selection of the construct.

Figure 9 The M protein was used as a model system to deliver fusion proteins to the surface of gram-positive bacteria. The surfaceexposed region of the molecule was genetically excised and replaced in frame with a foreign protein.

of the location of the surface-exposed and wall-associated regions of the fibrillar M protein (57,58). Thus, deleting the surface-exposed segment of the M molecule and replacing it in frame with the gene for a foreign protein (Fig. 9) [i.e., the E7 protein from human papillomavirus, consisting of 294 base pairs (101)], the fusion molecule could be presented on the bacterial cell surface and tightly anchored. Using this same strategy, a variety of protein antigens ranging from a few hundred to over 700 amino acids have now been successfully expressed on the surface of the human commensal S. gordonii [(101–104) and unpublished data]. To be certain that the expression of the recombinant molecule would be stable for many bacterial generations, the recipient S. gordonii was engineered so that the recombinant gene would be expressed from the chromosome under the control of an efficient resident promoter (100,101) (Figs. 10, 11). This strategy is one of few in which the gene in question is chromosomally expressed. In contrast, most other reported live vaccine vector systems regulate their genes from high expression plasmids. Translocation of the recombinant molecule to the surface is assured by inclusion of the signal sequence and a short segment of the N-terminal region of the carrier M protein. While the studies of Pozzi et al. (101) showed the feasibility to express a wide range of foreign proteins on the surface of gram-positive bacteria, an important question remained: Would this mode of delivery induce an immune response, particularly a mucosal response in animals colonized by the recombinant organism? To answer this question and to further verify the ability to deliver a diversity of proteins to the bacterial cell surface using this approach, King et al. elected to express a

Figure 11 Insertion of the fusion protein into the gram-positive chromosome. A cassette is inserted in the chromosome of Streptococcus gordonii in front of a high expression promoter selected at random. The cassette consists of a cat antibiotic reporter gene flanked by a short segment of the 50 end of the signal sequence of the M protein gene (S) and the 30 end by a short segment of the ermC marker. Since S. gordonii are naturally transformable, the linearized plasmid inserts the M protein fusion precisely in the chromosomal cassette in front of the promoter.

204 amino acid protein allergen from the white-faced hornet (Ag5.2) (105) on the surface of S. gordonii, using essentially the same methods described above (101,103) (Figs. 10, 11). These studies clearly showed that mice colonized with the recombinant S. gordonii expressing the allergen were able to mount not only a mucosal response, but a serum IgG and T-cell response specifically to the allergen expressed on the S. gordonii cell surface. Although still in development, the gram-positive commensal seems promising as a versatile live vector for vaccine delivery. Since the system induces both a mucosal and systemic immune response, it may be a more natural way of generating a protective response to a pathogen than systemic delivery alone. Although the animal studies indicate that this approach is feasible, human studies must be performed to determine if the same responses

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will be achieved. Early experiments show that when reintroduced into the human oral cavity, S. gordonii is capable of persisting for over two years (106). It remains to be seen if the recombinant will perform similarly and induce an immune response to the fusion protein expressed on its surface. Even though an immune response is in fact generated to commensal flora (107) even in humans ((108), unpublished data), it is not a clearing response. It may be expected, however, that the same would occur with the newly introduced recombinant strain. Nonpathogenic Lactococcus lactis that surface expresses the conserved C-repeat region of M protein has recently been used as a vaccine vector in place of S. gordonii (109). The authors found that mice immunized nasally with this live vaccine produced both sIgA and serum IgG directed to the M protein C-repeat fragment, whereas animals immunized subcutaneously produced serum IgG but not salivary IgA to the M protein. In protection studies, mice vaccinated nasally were protected against pharyngeal colonization following a nasal challenge with a heterologous Streptococcus pyogenes M serotype, while mice vaccinated subcutaneously were not protected against colonization. These results mirror the results with S. gordonii (103) and further emphasize the cross-protective effects of the mucosal approach using the conserved region of the M protein (84).

TOWARD VACCINE TRIALS WITH RECOMBINANT STREPTOCOCCUS GORDONII Convinced that the use of commensal bacteria as a vaccine delivery vehicle is a safe, effective, and inexpensive way to induce a mucosal response, a recombinant S. gordonii was prepared, which contained the C-terminal half of the M protein containing the exposed conserved region of the molecule. This segment was similar to that used successfully in the vaccinia virus experiments (see above) (94). S. gordonii expressing this conserved fragment of the M protein on its surface was used to successfully colonize all inoculated mice for up to 12 weeks. During this time, the animals raised a salivary IgA (Fig. 12) and serum IgG (not shown) response to the intact M protein. The

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amount of M protein–specific sIgA was up to 5% of the total IgA in the saliva of these animals. Experiments revealed that conserved region-specific IgA and IgG induced by this method do not cross-react with human heart tissue as determined by immunofluorescence assay (110). Only 25% of mice colonized with the recombinant S. gordonii and challenged with M14 streptococci had positive throat swabs or died five days after challenge, compared with 67% of control animals colonized with wild-type S. gordonii (Fig. 13). If proven successful, the commensal delivery system would be ideal for developing countries. Being a live vector, it would be easy to administer and not likely require additional doses. Also, since gram-positive bacteria are stable for long periods in the lyophilized state, a cold chain would not be required. Early studies show that when reintroduced into the human oral cavity, S. gordonii is capable of persisting for over two years, and is transmitted to other members of the family (106). For a developing country this factor could be ideal since rarely is the whole population able to be immunized. However, it remains to be seen if in humans the recombinant will induce a protective immune response to the M protein fragment expressed on its surface. Because of the uncertainties of using S. gordonii in humans as a live vaccine vector system, safety trials were performed utilizing this organism delivered orally and nasally to adult volunteers. The responses of 150 healthy volunteers to combined nasal and oral inoculation with approximately 1.5  109 CFU of SP204(1-1) were performed using an S. gordonii strain not bearing vaccine antigens (111). Strain SP204(1-1) was selected for resistance to streptomycin and 5-fluoro-2-deoxyuridine to distinguish it from indigenous flora. In two antibiotic treatment studies, serial cultures of nose, mouth, and saliva samples were performed from 120 subjects treated with azithromycin beginning five days after inoculation to determine whether SP204(1-1) could be rapidly eliminated in the event that vaccine safety concerns arise. A natural history study was performed to assess the time until spontaneous eradication in the remaining 30 subjects, who did not receive the antibiotic and who were

Figure 12 M protein–specific salivary immunoglobulin A in mice colonized with Streptococcus gordonii expressing the conserved region on the cell surface. Salivary samples were taken after pylocarpine induction and tested in enzyme-linked immunosorbent assay against the M protein.

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Figure 13 Mice were colonized with either recombinant Streptococcus gordonii expressing a conserved fragment of the M protein or wild-type S. gordonii and after 10 weeks were challenged orally with type 14 streptococci. Throat swabs were obtained five days after challenge to determine the extent of colonization. One CFU on blood agar plates was considered a positive swab. Twenty-five percent of the immunized mice had positive throat swabs or died within five days after challenge versus 67% of control animals that either died or had positive throat swabs.

monitored with repeated cultures for 14 weeks after inoculation. SP204(1-1) was generally well tolerated. Symptoms reported most often within five days of inoculation were nasal congestion (36%), headache (30%), and sore throat (19%). The strain was detected in cultures from 98% of subjects. A single dose of azithromycin eliminated colonization in 95% of subjects; all subjects receiving a five-day course of an antibiotic showed clearance by day 11. Without the antibiotic, 82% of subjects showed spontaneous eradication of the implanted strain within seven days, and all showed clearance by 35 days. The results of these clinical trials provide encouragement that the use of S. gordonii as a live mucosal vaccine vector is a feasible strategy.

RATIONALE FOR MULTIVALENT N-TERMINAL M PROTEIN–BASED VACCINES As well as being a major virulence factor of the organism and conferring resistance to phagocytosis, the M protein is a major protective antigen. Serum antibodies against M protein are opsonic and promote bactericidal killing of GAS that is mediated by polymorphonuclear leukocytes (53). Type-specific bactericidal antibodies that develop after natural infection correlate with protection against subsequent infection with the same serotype (53) and may persist for many years (47). These seminal observations, which were largely those of Lancefield, have served as the basis for M protein vaccine development for over 70 years. The overall goal of multivalent M protein–based vaccines is to approximate the type-specific protection induced following natural infection (47). Early studies by Fox and his coworkers clearly demonstrated the protective efficacy in humans of highly purified M protein vaccines (112,113). Volunteers that were immunized parenterally (112) or locally via the upper airway (113) were protected against challenge infections with the same serotype of GAS. M proteins contain protective (opsonic) epitopes and in some cases, human tissue cross-reactive epitopes (13). Because of the theoretical possibility of inducing autoantibodies, a challenge has been to separate the protective epitopes from the autoimmune epitopes so that vaccine preparations would contain only protective M protein peptides. Multiple studies from several laboratories have shown that the epitopes contained in the hypervariable, type-specific N-terminus of the M proteins (Fig. 3) evoke antibodies with the greatest bactericidal activity and are least likely to cross-react with host tissues (114).

In addition, the majority of the autoimmune epitopes of M proteins that have been identified are located in the middle of the mature M proteins and are distinct from the type-specific, protective epitopes (13,114). These observations have led some investigators to focus on the N-terminal type-specific peptides of M proteins for inclusion in multivalent vaccines (115–117). Synthetic and recombinant peptides as small as 10 amino acids have been shown to protect animals against subsequent challenge infections with homologous serotype of GAS (118). The finding that small peptides from the M proteins could evoke bactericidal antibodies that were not cross-reactive with human tissues prompted investigators to identify methods of designing and formulating vaccines that contained protective epitopes from multiple M serotypes. One approach has been to design fusion proteins that contain multiple N-terminal M peptides in tandem. The first of these was a trivalent synthetic peptide that was linked to an unrelated carrier (119). Subsequent vaccines were produced using recombinant techniques in which specific 50 regions of the emm genes were amplified by PCR and linked together in frame using unique restriction sites. Vaccines containing 4 (115), 6 (117), 8 (116), and now 26 (120) peptides from different M serotypes have been shown to evoke broadly opsonic antibodies in animals without evoking tissue cross-reactive antibodies. Clinical trials designed to assess the safety and immunogenicity of the hexavalent (117) and 26-valent (120) vaccines in adult volunteers have recently been completed.

PRECLINICAL EVALUATION OF A 26-VALENT M PROTEIN–BASED VACCINE Current epidemiological data indicate that the majority of GAS infections in the United States are caused by relatively few serotypes. Surveillance of invasive disease conducted by the U.S. Centers for Disease Control and Prevention has shown that during the years 1998 to 2000, 19 serotypes accounted for 84% of the total isolates (121). In ongoing studies to determine the serotype distribution of GAS recovered from pediatric cases of pharyngitis in the United States, it was shown that 16 different serotypes accounted for 97% of all cases of pharyngitis (122). These data indicate that a multivalent vaccine containing M protein fragments from a limited number of serotypes could potentially have a significant impact on the overall incidence of streptococcal infections within a population. Therefore, a

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Figure 14 Schematic diagram of the four recombinant fusion proteins contained in the 26-valent M protein–based vaccine. The number of amino acids contained in each M protein fragment is indicated below the M type. The four proteins are mixed in equimolar amounts and formulated with alum for intramuscular injection. M101 was formerly stNS5 and M114 was formerly st2967. The M13 is strain M13W, which has been newly designated M94.

26-valent vaccine was designed to include N-terminal M peptides from epidemiologically important serotypes of GAS. These include the serotypes commonly responsible for serious infections, uncomplicated pharyngitis in children, and the serotypes that are currently or historically associated with acute rheumatic fever. On the basis of this information, serotypes included in the 26-valent vaccine (Fig. 14) account for 78% of all invasive infections, 80% of all cases of uncomplicated pharyngitis, and theoretically 100% of all ‘‘rheumatogenic’’ serotypes. Also included in the vaccine is a new protective antigen of GAS (Spa) that is expressed by at least several serotypes (123). Thus, the 26-valent vaccine actually contains 27 distinct peptides. The 26-valent vaccine consists of four component fusion proteins (Fig. 14) that were mixed in equimolar ratios and formulated with alum to contain 400 mg of protein/dose. Three rabbits that received three intramuscular (IM) doses of the vaccine at 0, 4, and 16 weeks developed broadly opsonic antibodies that were not cross-reactive with human tissues (120). Antibody titers were determined by ELISA using serum obtained at 18 weeks against each of the purified recombinant dimeric peptide components of the vaccine. All preimmune titers were less than 200. Of the 81 immune serum titers determined (27 antigens  3 rabbits), 69 titers (85%) increased by fourfold or greater. The vaccine elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. To determine the functional activity of the M protein antibodies evoked by the 26-valent vaccine, in vitro opsonization and bactericidal tests were performed using each of the 26 serotypes of GAS. Opsonization assays were designed to determine the percentage of neutrophils that engulfed or were associated with streptococci after rotation in nonimmune human blood that contained either preimmune or immune rabbit serum. The preimmune sera from all three rabbits resulted in less than 10% opsonization of each of the 26 serotypes tested, indicating that the donor blood used for these assays did not contain antibodies against the test organ-

ism and that each organism was fully resistant to opsonization in nonimmune blood. Using 30% opsonization in the presence of immune serum as a positive threshold result, 18 of the 26 serotypes (69%) were opsonized by at least one of three immune rabbit sera. Bactericidal assays were also performed as an additional measure of the potential protective efficacy of the 26-valent vaccine (120). In these assays, each of the 26 serotypes of GAS was rotated in nonimmune blood for three hours in the presence of either preimmune or immune rabbit sera. In all experiments, the test mixture containing preimmune serum resulted in growth of the organisms to eight generations or more, again indicating that the human blood did not contain opsonic antibodies against the test strains and that each organism was fully resistant to bactericidal killing in nonimmune blood. Using 50% reduction in growth (percent killing) after the three-hour rotation in blood containing immune serum compared with the preimmune serum as a positive threshold, bactericidal activity was observed against 22 of the 26 serotypes tested. When the results of the opsonization and bactericidal assays were combined, 24 of the 26 serotypes (92%) tested were opsonized by the immune sera in one or both assays. These results showed that a highly complex 26-valent M protein–based vaccine was immunogenic in rabbits and evoked broadly opsonic antibodies against the vast majority of vaccine serotypes and did not evoke antibodies that were cross-reactive with human tissues. Phase I clinical trials to assess its safety and immunogenicity of the 26-valent vaccine in normal adult volunteers have recently been completed (see below).

CLINICAL TRIALS OF MULTIVALENT M PROTEIN–BASED VACCINES Several clinical trials that have recently been performed determine the safety and immunogenicity of multivalent GAS vaccines. The first phase I trial was under the direction of Karen Kotloff, MD and was conducted at the Center for Vaccine

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Development at the University of Maryland. This was a doseescalating study of a recombinant hexavalent fusion peptide (117) containing N-terminal M protein fragments from serotypes 1, 3, 5, 6, 19, and 24. Ascending doses (50, 100, and 200 mg) of vaccine (8–10 subjects/dose), formulated with aluminum hydroxide, were given IM in three spaced injections. One year of intensive follow-up revealed the vaccine to be well tolerated and there was no evidence of tissue cross-reactive antibodies or immunological complications. At the highest (200 mg) dose, vaccination elicited significant rises in geometric mean antibody levels to all six component M antigens by ELISA and to five of the six M types in the opsonophagocytosis assay. In addition, postvaccination increases in serum bactericidal activity of at least 30% were measured in 55% of assays. These results provided the first evidence in humans that a hybrid fusion protein represents a feasible strategy for evoking opsonic antibodies against multiple serotypes of GAS and represented a critical first step in the development of an impeded vaccine. The 26-valent vaccine described above has also now completed phases I and II clinical trials to determine its safety and immunogenicity in adult volunteers (124). These studies were under the direction of Scott Haperin, MD and Shelly McNeil, MD at the IWK Grace Health Center, Dalhousie University, Halifax, Canada. In the phase I component of this study, 30 adult volunteers received 400 mg of the 26-valent vaccine formulated with alum administered IM at 0, 4, and 16 weeks (124). Clinical and laboratory follow-up included assays for tissue cross-reactive antibodies, type-specific antibodies against the component peptides of the vaccine, and functional assays to detect bactericidal antibodies. The vaccine was found to be safe and well tolerated. Local reactogenicity was comparable to other alum-based vaccines in adults. None of the subjects had laboratory or clinical evidence of rheumatic fever or nephritis. None of the subjects developed tissue crossreactive antibodies. 26 of 27 of the vaccine peptides evoked a more than fourfold increase in the geometric mean antibody titer over baseline. Significant bactericidal activity was observed after immunization for all vaccine serotypes of GAS. A phase II study that included 70 adult volunteers has recently been completed and the results were similar to those obtained in the phase I trial (unpublished).

CONCLUSION Prevention of GAS infections and their sequelae with safe and effective vaccines has long been recognized as a promising goal. Recent advances in our understanding of the pathogenesis of infection, the molecular structure and function of protective antigens, and the availability of suitable animal models and in vitro correlates of protection now provide an ideal background for broad-based vaccine development efforts. Although GAS infections afflict all populations of the world, the overwhelming need for vaccines is in low-income countries where not only the incidence of streptococcal infections is high but also the morbidity and mortality resulting from complications such as RHD and invasive infections are significant. The dichotomy of disease burden that defines the need for GAS vaccines in lowincome countries versus economically advanced countries poses a significant barrier to vaccine development efforts, which in recent years have faltered. Public funding for basic and applied discovery is ongoing but the level of support is insufficient for the required translational, and clinical studies

and at present commercial interest is very low. Further development of GAS vaccines will require a commitment of resources from other sources to identify the most promising antigens, assuage safety concerns, and to move vaccine candidates along the path of clinical development leading to large-scale efficacy trials and their eventual deployment to high-risk populations.

ACKNOWLEDGMENTS The work of Dr. Cleary is supported in part by USPHS Grant AI20016 and a grant from Wyeth Pharmaceuticals. The work of Dr. Dale is supported in part by USPHS Grant AI10085 and AI060592 and research funds from the Department of Veterans Affairs. The work of Dr. Fischetti is supported in part by USPHS Gant AI11822. The work of Drs. Batzloff, McCarthy, and Good is supported in part by grants from the NHMRC (Australia), National Heart Foundation (Australia), The Prince Charles Hospital Research Foundation (Australia), and the National Institutes of Health (United States). Dr Batzloff is a recipient of a Research Fellowship from the National Heart Foundation (Australia).

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Chapter 62: Group A Streptococcal Infections and Rheumatic Fever 97. Schneewind O, Mihaylova-Petkov D, Model P. Cell wall sorting signals in surface proteins of gram-positive bacteria. EMBO J 1993; 12:4803–4811. 98. Cole RM, Hahn JJ. Cell wall replication in Streptococcus pyogenes. Science 1962; 135:722–724. 99. Fischetti VA, Pancholi V, Schneewind O. Conservation of a hexapeptide sequence in the anchor region of surface proteins of gram-positive cocci. Mol Microbiol 1990; 4:1603. 100. Pozzi G, Oggioni MR, Manganelli R, et al. Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion. Res Microbiol 1992; 143:449–457. 101. Pozzi G, Contorni M, Oggioni MR, et al. Delivery and expression of a heterologous antigen on the surface of streptococci. Infect Immun 1992; 60:1902–1907. 102. Manca F, Habeshaw J, Dalgleish A. The naive repertoire of human T helper cells specific for gp120, the envelope glycoprotein of HIV. J Immunol 1991; 146:1964–1971. 103. Medaglini D, Pozzi G, King TP, et al. Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc Natl Acad Sci U S A 1995; 92:6868–6872. 104. Piard JC, Hautefort I, Fischetti VA, et al. Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria. J Bacteriol 1997; 179:3068–3072. 105. King TP, Kochoumian L, Lam T. Immunochemical observations of antigen 5, a major venom allergen of hornets, yellowjackets and wasps. Mol Immunol 1987; 24:857–864. 106. Svanberg M, Westergren G. Persistence and spread of the orallyimplanted bacterium Streptococcus sanguis between persons. Arch Oral Biol 1986; 31:1–4. 107. Shroff KE, Meslin K, Cebra JJ. Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut. Infect Immun 1995; 63:3904–3913. 108. Dougan G. 1993 Colworth Prize Lecture. The molecular basis for the virulence of bacterial pathogens: implications for oral vaccine development. Microbiology 1994; 140(pt 2):215–224. 109. Mannam P, Jones KF, Geller BL. Mucosal vaccine made from live, recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes. Infect Immun 2004; 72:3444–3450. 110. Zabriskie JB, Hsu KC, Seegal BC. Heart-reactive antibody associated with rheumatic fever: characterization and diagnostic significance. Clin Exp Immunol 1970; 7:147–159.

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111. Kotloff KL, Wasserman SS, Jones KF, et al. Clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a Streptococcus gordonii carrier strain intended for future use as a group A streptococcus vaccine. Infect Immun 2005; 73:2360–2366. 112. Fox EN, Waldman RH, Wittner MK, et al. Protective study with a group A streptococcal M protein vaccine. Infectivity challenge of human volunteers. J Clin Invest 1973; 52:1885–1892. 113. Polly SM, Waldman RH, High P, et al. Protective studies with a group A streptococcal M protein vaccine. II. Challenge of volunteers after local immunization in the upper respiratory tract. J Infect Dis 1975; 131:217–224. 114. Dale JB. Group A streptococcal vaccines. Infect Dis Clin North Am 1999; 13:227–243, viii. 115. Dale JB, Chiang EY, Lederer JW. Recombinant tetravalent group A streptococcal M protein vaccine. J Immunol 1993; 151: 2188–2194. 116. Dale JB, Simmons M, Chiang EC, et al. Recombinant, octavalent group A streptococcal M protein vaccine. Vaccine 1996; 14: 944–948. 117. Dale JB. Multivalent group A streptococcal vaccine designed to optimize the immunogenicity of six tandem M protein fragments. Vaccine 1999; 17:193–200. 118. Dale JB, Chiang EC. Intranasal immunization with recombinant group a streptococcal M protein fragment fused to the B subunit of Escherichia coli labile toxin protects mice against systemic challenge infections. J Infect Dis 1995; 171:1038–1041. 119. Beachey EH, Seyer JM, Dale JB. Protective immunogenicity and T lymphocyte specificity of a trivalent hybrid peptide containing NH2-terminal sequences of types 5, 6 and 24 M proteins synthesized in tandem. J Exp Med 1987; 166:647–656. 120. Hu M, Walls M, Stroop S, et al. Immunogenicity of a 26-Valent Group A Streptococcal Vaccine. Infect Immun 2002; 70: 2171–2177. 121. Schuchat A, Hilger T, Zell E, et al. Active bacterial core surveillance of the emerging infections program network. Emerg Infect Dis 2001; 7:92–99. 122. Shulman S, Tanz R, Kabat W, et al. U.S. Nationwide streptococcal pharyngitis serotype surveillance. Abstract of the Infectious Diseases Society of America 39th annual meeting, San Francisco, CA 2001:87 (abstr no 277). 123. Dale JB, Chiang EY, Liu SY, et al. New protective antigen of group A streptococci. J Clin Invest 1999; 103:1261–1268. 124. McNeil SA, Halperin SA, Langley JM, et al. Safety and immunogenicity of 26-valent group a streptococcus vaccine in healthy adult volunteers. Clin Infect Dis 2005; 41:1114–1122.

63 Vaccines Against Group B Streptococcus Morven S. Edwards Section of Infectious Diseases, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, U.S.A.

Carol J. Baker Section of Infectious Diseases, Departments of Pediatrics and of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, U.S.A.

Lawrence C. Paoletti Brigham and Womens’ Hospital, Harvard Medical School, Channing Laboratory, Boston, Massachusetts, U.S.A.

GROUP B STREPTOCOCCAL DISEASE BURDEN Newborn and Young Infants Group B Streptococcus (GBS) has been a major cause of serious bacterial infection in neonates and young infants since the 1970s. Use of empirical antibiotic therapy for suspected infection based on delineated risk factors reduced case fatality rates and improved outcomes for infants with invasive GBS disease, but disease incidence was stable until the mid-1990s when clinical trial-based recommendations demonstrated the efficacy of maternal intrapartum antibiotic prophylaxis (IAP) with intravenous penicillin G or ampicillin in preventing early-onset (age <7 days) disease. The incidence of early-onset disease declined significantly in association with implementation of consensus IAP guidelines endorsed in 1996 by the American Academy of Pediatrics, the American College of Obstetricians and Gynecologists, and the Centers for Disease Control and Prevention, and revised guidelines endorsed in 2002 that now recommend universal screening of pregnant women for GBS rectovaginal colonization and use of IAP for all GBS carriers (1). Active, multistate, population-based surveillance documented a 65% decrease in incidence from 1.7 cases per 1000 live births in 1993 to 0.6 per 1000 in 1998 (2). Early-onset disease incidence declined further to 0.33 cases per 1000 live births during 2003 to 2005 (3). Contemporary appraisal of the epidemiology of neonatal disease has affirmed that low birth weight and black ethnicity continue to be associated with enhanced risk for early-onset disease (3), and these are the populations with substantially less incidence reduction in the IAP era. The predominant serotypes of GBS currently causing neonatal and young infant disease are Ia, III, and V; these account for *80% to 85% of invasive infections identified in a racially and ethnically diverse cohort from metropolitan areas in the United States (4). Early-onset infection most commonly manifests as bacteremia, but *6% to 7% of infants present with meningitis and a similar proportion with pneumonia. Maternal IAP has not decreased the number of late-onset (7 to 89 days of age) infant infections; the incidence persists at 0.3 to 0.4 cases per 1000 live

births (3). Approximately one-quarter of infants with late-onset GBS disease present with meningitis, and a substantial number of survivors (estimated at 20%) have neurological sequelae. Despite the widespread implementation of maternal IAP in the United States, it is projected 1425 infants 0 to 6 days of age and 1375 infants 7 to 89 days of age will develop invasive GBS disease yearly, and that *80 of these will have fatal infection (5).

Children GBS disease in children 90 days to 14 years of age comprises a small but important portion of the total disease burden. A multistate active bacterial surveillance group reported that 2% of cases occurred in this age group, as opposed to 28% in the 0 to 90 days of age group (2). One-half of these patients were infants younger than six months of age. The survival of extremely low birth weight infants, particularly those requiring long-term hospitalization, has extended the age of susceptibility for young infants beyond the original definition for occurrence of late-onset disease ending at age three months. Many of these late, late-onset infections manifest as bacteremia, and a benign outcome is typical. Children older than six months of age often have underlying condition(s) predisposing to invasive infection, such as structural heart or central nervous system defects (6). The mortality rate for GBS disease in childhood is about 9%, or nearly twice that for neonates.

Pregnant Women Lower vaginal or rectal colonization with GBS is a risk factor for invasive infection in pregnant women and their neonates. The risk of intra-amniotic infection or chorioamnionitis is higher for women with high (>105 CFU/mL) than for those with low density GBS vaginal colonization (7). The advent of maternal IAP has impacted invasive GBS disease, primarily bacteremia with or without endometritis or chorioamnionitis, among pregnant women, with a decline in incidence from 0.29 cases per 1000 deliveries in 1993 to 0.12 per 1000 during

Chapter 63: Vaccines Against Group B Streptococcus

2003 to 2005 (2,3). Substantial morbidity persists from bacteremia, chorioamnionitis, endometritis, and septic abortion attributable to GBS during pregnancy. Of pregnancies with known outcomes, 62% resulted in abortion or stillbirth, 6% resulted in neonatal death, 2% had infants who had nonfatal GBS disease, and 30% had infants who remained well (3).

Nonpregnant Adults The decline in neonatal GBS incidence contrasts with a recent two to fourfold increase in the incidence of invasive GBS disease in nonpregnant adults (8,9). More than three-quarters of the total cases of invasive GBS disease in the United States affect adults, and most of these infections are unrelated to pregnancy (5). Disease rates rise with advancing age; in two reports, adults 65 years of age or older had GBS infection rates of 11.9 and 28.3 per 100,000 population, respectively (10,11). Nursing home residents are at particularly high risk. The age-adjusted annual incidence of GBS per 100,000 population among those 65 years of age and older is 72.3 for nursing home residents and 17.5 for community residents (relative risk, 4.1) (12). Elderly adults are projected to account for *8000 cases of invasive GBS infections in the United States yearly, or more than one-half of the total adult cases, and for more than one-half of the total deaths attributable to invasive GBS infections (5). Most adults have underlying medical conditions predisposing to invasive GBS infection (8,13). Diabetes mellitus is a risk factor for 20% to 40% of these patients. Cirrhosis or other chronic liver disease, breast cancer, neurogenic bladder, and decubitus ulcer also are associated with significantly increased risk (10). The aging of the U.S. population, together with improving treatments for chronic diseases, suggest that GBS disease in nonpregnant adults will pose an ongoing and increasing proportion of the disease burden.

CLINICAL TRIALS WITH VACCINES FOR GBS This following discussion is arranged chronologically from the earliest GBS candidate vaccines, purified capsular polysaccharides (CPS), to the development CPS-protein conjugate vaccine constructs. Clinical trials began with monovalent CPS for serotype III, followed by those for type Ia and II, based on the burden of perinatal disease attributable to these types in the 1970s and early 1980s. With the more recent availability of GBS conjugate vaccines, types Ib and V vaccines have also been evaluated in healthy adults. Additional studies have included testing of an adjuvant, effect of a second dose on the immune response to type III conjugate vaccine, and of a bivalent type II/ III conjugate vaccine. Most of these candidate GBS vaccines were developed and prepared at the Channing Laboratory, Harvard Medical School, Boston, Massachusetts, U.S.A.

GBS Capsular Polysaccharide Vaccines A protective role for antibodies against the CPS of GBS was suggested by Lancefield’s observations in a mouse model of lethal infection. A protective role for CPS-specific antibodies in human disease was reported by Baker and Kasper (14), who found that neonates at risk for invasive CPS type III GBS disease were born to women with a low concentration of III CPS-specific immunoglobulin (Ig)G in their sera at delivery. This association was confirmed for types Ia and Ib (15,16). Antibodies against specific CPS promoted opsonization of homologous capsular types for phagocytosis and killing by

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human neutrophils in vitro, and protected against lethal infection in experimental animal models. Initial characterization of CPS antigens of GBS by Lancefield employed hot hydrochloric acid extraction from whole organisms. This method degraded the later discovered terminal sialic acid moieties, a critical structure for type III GBS because these moieties of the repeating side chain of the CPS dictate the tertiary structure of the molecule that defines the protective epitope. ‘‘Complete’’ or ‘‘native’’ CPS of types Ia, II, and III GBS as candidate vaccines, though well tolerated in healthy adults, were inconsistently immunogenic. A majority of adults had low preimmunization concentrations of CPS-specific antibodies in sera, and these predicted a poor response to immunization. In adults with low preimmunization concentrations of CPSspecific antibodies, 88% developed significant immune responses to type II CPS, but for Ia and III CPS, the responses were 40% and 60%, respectively (17). Forty healthy pregnant women were immunized with purified III CPS at a mean of 31 weeks of gestation. Among the 25 women who responded to the native CPS vaccine, 90% had infants with predicted protective levels of III CPS-specific IgG in their cord sera, and the majority of sera from these infants promoted phagocytosis and killing in vitro of type III GBS through age two months (the latest age tested). These observations supported the concept that maternal immunization could elicit potentially protective CPS-specific, IgG class antibodies that would be passively transferred neonates and prevent GBS disease during the first three months of life (18).

GBS Type III CPS-Protein Conjugate Vaccine The initial emphasis of conjugate vaccine development was based on the high proportion of disease in neonates and young infants caused by III GBS (currently still estimated at 60%), and data indicating that antibodies directed at the III CPS were protective against infant infection (14,15). Strategies to improve immunogenicity included the use of enzymatically derived oligosaccharides and native polysaccharides as haptens. Conjugate vaccines prepared with III CPS oligosaccharides were highly immunogenic in animals, and useful for study of immune responses to a polymer possessing conformationally dependent epitopes, but methods to derive III CPS oligosaccharides and the end-linking chemistry were inefficient, so these vaccines could not be made on large scale (19,20). Conjugate vaccines using native type III CPS and III–tetanus toxoid (TT) with two different coupling chemistries (21,22) resulted in vaccines that were immunogenic in animals, elicited antibodies in adult mice that protected pups from experimental challenge, and produced functionally active III CPS-specific IgG in mice, rabbits, and baboons (22–24). To date, all GBS conjugate vaccines used in clinical trials have been developed using reductive amination coupling chemistry (22,25). Issues of conjugate size, polysaccharide size, and degree of polysaccharide-protein cross-linking influence the immunogenicity and protective efficacy of III-TT conjugate vaccines (26). These structural properties have been considerations in the vaccines formulated to date. The first GBS conjugate vaccine for clinical trials was created using purified III CPS with a Mr of approximately 200,000; aldehydes were formed on 26% of sialic acid residues before coupling to monomeric TT. This III-TT conjugate vaccine, prepared at the Channing Laboratory, Boston, under good manufacturing practices (GMP) conditions, was 56%

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Table 1 Response to GBS Conjugate Vaccines in Healthy Adults Eight Weeks Post Immunization GBS vaccine (CPS dose in mg) III-TT (58) III-TT (14.5) III-TT (3.6) Ia-TT (60) Ia-TT (15) Ia-TT (3.75) Ib-TT (63) Ib-TT (15.75) Ib-TT (3.94) II-TT (57) II-TT (14.3) II-TT (3.56) V-TT (50) V-CRM (50) V-TT (38.5) V-TT (9.6) V-TT (2.4) II/III-TT (3.56/12.5)

Concentration (mg/mL) of CPS-specific IgG

Number of subjects

Geometric mean

Range

95% CI

% with
4-fold increase in CPS-specific IgG by ELISA

30 15 15 30 15 15 30 15 15 30 13 15 15 15 15 15 15 25

4.53 2.72 1.1 26.2 18.3 1.9 12.9 11.1 2.9 34.3 29.5 11.2 8.2 5.7 11.1 5.5 5.1 13.1/6.9

1.92–10.70 0.95–7.76 0.4–3.02 13.0–52.9 6.0–55.4 0.7–5.4 5.2–32.0 3.3–37.0 1.2–7.1 18.7–62.4 13.9–62.4 6.1–20.8 3.2–21.1 2.2–14.9 3.8–31.9 1.5–20.5 1.3–20.0 5.6–30.6; 3.2–14.9

0.07–325.12 0.19–98.30 0.06–28.99 0.5–497.9 0.7–469.4 0.1–152.8 0.1–443.0 0.2–188.5 0.1–71.0 0.4–535 1.9–176 0.9–83.1 0.6–125 0.2–435 0.5–133 0.2–474 0.06–171 0.4–571; 0.1–237

97 87 64 93 80 80 78 80 47 97 100 87 93 93 100 93 80 88/>90

Abbreviations: GBS, group B Streptococcus; CPS, capsular polysaccharides; Ig, immunoglobulin; TT, tetanus toxoid. Source: From Refs. 27, 28, 30, 38, 39, and 41.

carbohydrate and 44% protein (27). As with all GBS conjugate vaccines, this vaccine passed tests for general safety, microbial sterility, and pyrogenicity required by the Food and Drug Administration. Sixty healthy nonpregnant women 18 to 40 years of age were randomized to receive a single intramuscular injection of III-TT conjugate at a dose containing of 58, 14.5 or 3.6 mg of III CPS or the unconjugated III CPS without adjuvant at a dose of 50 mg. Each vaccine and dose of III-TT conjugate was well tolerated. The majority of women experienced no local symptoms or only injection site tenderness and one-quarter had soreness with arm movement. No more than 27% developed redness or swelling at the injection site, and when this occurred, it was mild (<3 cm) in the majority, resolving within 72 hours after vaccine administration. The geometric mean concentration (GMC) of III CPSspecific IgG in sera from the 30 recipients of the 58-mg dose of III-TT rose from 0.09 mg/mL before to 4.89 mg/mL two weeks after immunization, remained relatively unchanged eight weeks after immunization (Table 1), and was 3.02 mg/mL 18 weeks later. The immunogenicity of III-TT was dosedependent. Proportional response rates (
4-fold rises) eight weeks after immunization were 97% in recipients of the highest dose and 64% in those receiving the 3.6 mg dose. Women receiving unconjugated III CPS had significantly inferior immune responses (data not shown). A positive correlation (r ¼ 0.7) was found between complement-dependent killing of type III GBS by healthy adult neutrophils and the concentration of III CPS-specific IgG in four weeks postimmunization sera. The functional activity of III CPS-specific IgG was also shown by protection studies in mice. A pooled standard human reference serum (SHRS III) from five III-TT recipients (83.5 mg of III CPSspecific IgG) was administered intraperitoneally to pregnant dams. Thirty (73%) of 41 pups born to dams given SHRS III survived a usually lethal challenge of type III GBS, whereas the 11 pups born to a dam given normal human serum died (27). Thus, these phase 1 and 2 trials demonstrated, in concept, the potential for GBS conjugate vaccines to prevent GBS disease in neonates and young infants through maternal immunization.

GBS Types Ia and Ib CPS Conjugate Vaccines Two variables influence the number of aldehydes formed on GBS CPS, the size of the CPS (and thus the number of sialic acid residues) and the amount of sodium periodate added to the sialic acid oxidation reaction. Unlike other GBS CPS, the type Ia and Ib have a Mr > 800,000. When the number of aldehydes created on these two GBS polysaccharides exceeded *40%, the reductive amination coupling with TT resulted in the formation of insoluble gels, so that a different approach for these glycoconjugate candidates was required. Preclinical lots of soluble type Ia-TT and Ib-TT conjugate vaccines were immunogenic in mice and rabbits, and prevented neonatal GBS disease in the maternal vaccination-neonatal mouse model (28). On the basis of these preclinical data, type Ia-TT and Ib-TT conjugate vaccines were prepared with 25% and 9%, respectively, of sialic acid oxidation. Both vaccines were composed of 66% and 34% carbohydrate and protein, respectively. Ia-TT conjugate contained 60 mg of CPS/0.5 mL dose, while Ib-TT conjugate contained 63 mg of CPS/0.5 mL dose. One hundred and twenty healthy, nonpregnant women, ages 18 to 40 years, were randomized to receive a single intramuscular dose of either type Ia-TT or type Ib-TT conjugate vaccine (28). Thirty women received Ia-TT or Ib-TT at dosages of either 60 mg (type Ia) or 63 mg (type Ib) of the polysaccharide component, and groups of 15 women received one of two 4-fold decreasing doses of each vaccine. The vaccines were well tolerated; no serious adverse effects were observed. Most vaccine recipients had only mild injection site tenderness or no local symptoms or signs. Before vaccination, sera from each of the vaccine dose groups had low concentrations of Ia or Ib CPS-specific IgG ( 0.6 mg/mL) (28). Among recipients of the 60 mg Ia-TT conjugate vaccine dose, the Ia CPS-specific IgG GMC increased to 21.6 mg/mL two weeks after vaccination and peaked at 26.2 mg/mL at eight weeks (Table 1). The 15 mg dose of Ia-TT conjugate evoked a GMC that did not differ significantly from that in the sera from 60 mg dose recipients, whereas Ia CPSspecific IgG evoked by the lowest dose (3.75 mg) was

Chapter 63: Vaccines Against Group B Streptococcus

significantly lower. Ninety-three percent of the 30 women who received the 60 mg dose of Ia-TT conjugate and 80% of those given the 15 mg dose had
4-fold increases in serum Ia CPSspecific IgG at eight weeks after vaccination (Table 1). Recipients of the higher doses of the Ib-TT conjugate vaccine had immune responses that did not differ significantly from each other, but evoked a significantly higher GMC of Ib CPS-specific IgG than did the 3.94 mg dose. Antibody responses to the two higher doses peaked four to eight weeks after vaccination, and changed minimally in the ensuing 18 weeks. In an analysis two years after vaccination, the Ib CPS-specific IgG GMC in sera from 13 recipients of the 63 mg dose was 10.7 mg/mL (range 0.26–147.7), indicating that this immune response was durable. When immunogenicity was analyzed by
4-fold increases in Ib CPS-specific IgG, 78% and 80% of recipients of the 63 mg and the 15.75 mg doses, respectively, achieved these fold rises. A functional correlate was shown by opsonophagocytic assay between the concentration of Ia and Ib CPS-specific IgG and neutrophil-mediated killing by sera from recipients of type Ia (r ¼ 0.65) and type Ib (r ¼ 0.80) conjugate vaccines. Taken together, the phase 1 trials with GBS types Ia, Ib, and III documented consistently low reactogenicity, substantial immunogenicity, in vitro function of vaccine-induced specific antibodies, and durable immune responses.

GBS Type II CPS Conjugate Vaccine GBS type II CPS contains sialic acid as one of two monosaccharide side chains in its seven-sugar repeating unit (29). The relationship between the amount of sodium periodate added and the formation of aldehydes on sialic acid residues was not direct, as observed with GBS types Ia, Ib, and III, possibly because of the proximity of sialic acid residues on the II CPS to the backbone sugars. These structural features required use of a higher concentration of sodium periodate to create a sufficient number of aldehydes on the type II CPS for coupling to a protein carrier. To determine the degree of sialic acid oxidation that would yield immunogenic and efficacious conjugate vaccines, preclinical lots of II CPS-TT conjugate vaccines were prepared with CPS containing 31%, 57%, and 73% sialic acid oxidation. Immunogenicity and protective efficacy indicated that the II CPS prepared with 57% oxidation was the most consistently immunogenic and protective in mice, results that prompted the preparation of II-TT conjugate vaccine for clinical trails with CPS containing 35% sialic acid oxidation (30).

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GBS II-TT conjugate was composed of 51% carbohydrate and 49% protein. The decision to bottle the II-TT vaccine as a lyophilized multidose preparation was based on observations that lyophilized GBS oligosaccharide conjugates retained antigenic properties (31), and was a more conventional manner of bottling vaccines. The II-TT conjugate lyophilized vaccine used sucrose as the excipient to serve as a stabilizer and to add bulk to the mixture. When the multidose vial was reconstituted with phosphate-buffered saline containing 0.01% thimerosal, each 0.5-mL dose contained 57 mg of conjugate II CPS and 55 mg of TT. This vaccine was 95% protective in a mouse maternal vaccination-neonatal challenge model of GBS disease, a measure of GBS vaccine potency. Potency persisted four years after it was vialed (31), demonstrating the long-term stability of a lyophilized GBS conjugate vaccine. The type GBS II-TT conjugate vaccine was well tolerated when given as a single intramuscular dose to 60 healthy, nonpregnant women. Most vaccine recipients experienced only mild or moderate pain at the injection sites. One subject had mild systemic symptoms probably related to immunization. Before immunization, the GMC of II CPS-specific IgG in sera from the three groups of women receiving fourfold decreasing vaccine doses was low ( 0.5 mg/mL) (30). In sera from the 30 recipients of the highest II-TT dose (57 mg), the II CPS-specific IgG GMC increased to a peak of 47.1 mg/mL two weeks after immunization. Eight weeks after immunization, the II CPS-specific IgG GMC ranged from 11.2 to 34.3 mg/mL (Table 1), each representing a concentration that would ensure that adequate CPS-specific IgG would be available for placental transport. The II CPS-specific IgG responses were durable when assessed at 26 and 104 weeks after vaccination. Interestingly, the II-TT vaccine elicited substantial II-specific IgM and IgA responses. For example, eight weeks after immunization, the 3.56 mg dose elicited a II CPS-specific IgM GMC of 5.5 mg/mL (95% CI 3.0–10.2) and a IgA specific GMC of 0.8 mg/mL (95% CI 0.4–1.5) (Table 2). The immunological explanation for this diversity of Ig isotype distribution in response to II-TT conjugate compared with type Ia, Ib, and III conjugate vaccines requires further study. When functional activity of sera from adults immunized with II-TT conjugate vaccine to promote ingestion and killing of an opsonoresistant type II GBS strain before and four weeks later was studied, a significant increase was observed. The concentration of II CPS-specific IgG correlated with opsonophagocytic activity of sera from vaccine recipients against type II GBS.

Table 2 Isotype-Specific Immunogenicity of Type II and Type V GBS Conjugate Vaccines in Healthy Adults Eight Weeks After Immunization Concentration (mg/mL) of CPS-specific antibody Vaccine (mg)

Number of subjects

II-TT (3.56)

15

V-TT (50)

V-CRM (50)

ELISA

Geometric mean

Range

95% CI

IgG IgM IgA

11.2 5.5 0.8

6.1–20.8 3.0–10.2 0.4–1.5

0.9–83.1 1.2–26.6 0.3–2.8

15

IgG IgM IgA

8.2 17.8 4.8

3.2–21.1 7.0–45.6 1.8–12.4

0.6–125 0.6–277 0.4–97.9

15

IgG IgM IgA

5.7 27.8 7.2

2.2–14.9 14.8–52.4 3.6–14.2

0.2–435 4.8–182 1.2–138

Abbreviations: Ig, immunoglobulin; TT, tetanus toxoid. Source: From Refs. 30 and 38.

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GBS Type V CPS Conjugate Vaccines Epidemiological studies during the 1970s and 1980s documented that virtually all-invasive GBS infections in the United States were caused by types Ia, Ib, II, and III. In the early 1990s, a new GBS serotype emerged (32–34), which had been described by Jelı´nkova´ and Motlova´ (35) in Prague in the mid-1980s. With type V GBS adding to the perinatal disease burden, its CPS was purified and structurally characterized (36). Type V CPS is structurally unique, but like the other GBS CPS types, possesses a side chain that terminates with sialic acid so reductive amination could also be utilized to create a type V conjugate vaccine (36). GBS type V CPS conjugate vaccines were prepared with TT as the carrier protein and tested in rabbits and mice. Unlike the uncoupled V CPS, the V-TT conjugate induced type-specific antibodies in rabbits that opsonized the type V strain in vitro for killing by human polymorphonuclear leukocytes (37). Like the other GBS serotypes, type V-TT conjugate vaccine was also superior to uncoupled type V CPS in protective efficacy against a lethal type V challenge to neonatal mice born to vaccinated dams (37). Two lots of GBS type V conjugate vaccine were prepared for use in phase 1 clinical trials. The first used TT as the carrier protein and was 76% carbohydrate and 14% protein, and the second cross-reactive material 197 (CRM197, kindly provided by R. Rappuoli, IRIS, Siena, Italy), and was 73% carbohydrate and 17% protein. Both conjugate vaccines were prepared with purified type V CPS (Mr 173,000) that had 18% of its sialic acid residues oxidated (38). Like GBS II-TT conjugate, these type V conjugate vaccines were prepared as multidose lyophilized preparations with sucrose as the excipient. Thirty healthy, nonpregnant women, aged 18 to 45 years, received one of these two type V conjugates. Sera from both groups of women had similarly low preimmunization concentrations of V CPS-specific IgG (GMC  0.2 mg/mL), and both groups developed significant increases at each postvaccination interval through 26 weeks (Table 1). Type V-TT conjugate vaccine recipients had somewhat higher V CPS-specific IgG concentrations in their sera following immunization compared to the V-CRM group [e.g., GMC of 8.2 vs. 5.7 mg/mL at eight weeks (Table 1)], but the differences were not statistically significant. Postvaccination sera promoted opsonophagocytosis and killing in vitro. Fourfold or greater increases in V CPSspecific IgG were observed in 93% of subjects in each vaccine group, and these persisted in 85% (V-TT) to 93% (V-CRM) of women two years later, indicating good durability of vaccineinduced antibodies. A dose-escalating trial conducted in healthy adults with V-TT conjugate vaccine suggested that a low dose of V-TT (*2.4 mg of V CPS) could optimize the immune response to type V conjugate (Table 1) (39). V-TT conjugate vaccine has also been evaluated in a phase 1 study, and found to be safe and immunogenic in healthy adults 65 to 85 years of age, suggesting the potential for prevention of invasive GBS disease in the elderly through immunization (40). By contrast to results using type Ia, Ib, and III-TT conjugate vaccines but similar to those with II-TT conjugate vaccine, a substantial proportion of the V CPS-specific antibodies produced after immunization with either V-TT or V-CRM were of isotypes other than IgG. Immunization induced a brisk V CPSspecific IgM and IgA response, and the V CPS-specific IgM GMCs exceeded those for IgG (Table 2). Type V conjugate vaccine recipients had significant increases in V CPS-specific IgM and IgA four and eight weeks after immunization, which persisted for two years. Recipients of V-CRM conjugate had

substantially greater increases in V CPS-specific IgA than those who received V-TT conjugate, but this apparent difference was significant only at the two-week interval.

GBS Type II-TT/III-TT Bivalent Vaccine A clinical trial was designed to determine whether administration of two monovalent GBS conjugate vaccines, II-TT and IIITT, stimulated immune responses comparable to those elicited by either of the monovalent vaccines (41). A phase 2 randomized, double-blinded trial in 75 healthy adults between the age of 18 and 45 years was conducted. One group received II-TT (3.56 mg CPS/3.4 mg TT), another III-TT (12.5 mg CPS/16.1 mg TT), and the third the bivalent II-TT/III-TT (3.56 II mg CPS/ 12.5 mg/III CPS/19.5 mg TT) each as a single 0.5 mL intramuscular dose. The choice of the concentration for each antigen in the bivalent vaccine was based on our earlier finding that at these doses in nearly 90% of the volunteers receiving a single conjugate elicited a
4-fold increase in the CPS-specific IgG. Each vaccine was well tolerated. Bivalent II/III-TT conjugate elicited increases in II or III CPS-specific IgG that were similar to those stimulated by the monovalent vaccines. A more than fourfold increase in II CPS-specific IgG was noted in more than 80% of sera from II-TT or bivalent vaccine recipients, and in >90% of sera from subjects given III-TT vaccine. Unexpectedly, 25% of III-TT subjects also developed more than fourfold rises in II CPS-specific IgG, indicating immunological cross-reactivity between the two structurally similar disaccharides in these two CPS. No immune interference from combining these vaccines was observed, results suggesting the feasibility of developing a multivalent GBS CPS-protein conjugate vaccine (41).

Booster Dose of GBS Type III Conjugate Vaccine To move toward the goal of evaluating the safety and immunogenicity of GBS conjugate vaccines in pregnant women, a GBS type III-TT vaccine prepared under GMP conditions was needed to satisfy IND requirements by the Food and Drug Administration. In 1995, GMP lots of III-TT vaccine were prepared at the Salk Institute, Swiftwater, Pennsylvania, U.S.A., in multidose vials containing sucrose excipient. Approximately 30% of the sialic acid residues of III CPS were periodate oxidized to create reactive aldehydes as sites for coupling to TT (61% carbohydrate and 39% protein by weight for the first lot and 44% carbohydrate and 56% protein by weight for the second). The second vaccine lot was used in a trial designed to evaluate the potential of a second dose of vaccine on immunogenicity (42). Thirty-six healthy men and women, aged 18 to 50 years, vaccinated previously with III-TT were given a second 12.5 mg dose of III-TT conjugate 21 months later. The vaccine was well tolerated, and there was no increase in the frequency with which local or systemic responses were observed. Four weeks after this second vaccine dose, the GMC of III CPS-specific IgG (8.4 mg/mL) was similar to that measured after the first dose (8.8 mg/mL), suggesting lack of a ‘‘booster’’ effect. However, a subset of 22% of the subjects who had undetectable III CPSspecific IgG levels (<0.5 mg/mL) before the first dose of III-TT conjugate exhibited a ‘‘booster’’ response to the second dose. This was defined as a
4-fold higher GMC of III CPS-specific IgG than after the initial vaccine dose. Thus, a second dose of GBS CPS conjugate vaccine may be required for adults previously ‘‘naı¨ve’’ or ‘‘unprimed’’ to type III GBS CPS or to a related antigen. A second vaccine dose might also be useful to

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restore the initial peak III CPS-specific IgG serum levels in those previously responding to vaccination.

Alum as an Adjuvant Antibody responses to GBS conjugate vaccines in healthy adults peak four to eight weeks after administration of a single dose. In an effort to improve the magnitude and proportion of the responses to GBS conjugate vaccines, an adjuvant trial was conducted using III-TT conjugate. The vaccine was adsorbed to aluminum hydroxide gel in 0.9% saline (alum) prepared by the Massachusetts Public Health Laboratory, Jamaica Plain, Massachusetts, U.S.A., under GMP conditions using Alhydrogel 1.3% (aluminum hydroxide gel adjuvant) manufactured by Superfos Biosector a/s, Vedbaek, Denmark (43). The rationale for choosing alum was based on its widespread use in human vaccines and its ability to improve the immunogenicity of GBS conjugate vaccines in mice (43) and baboons (23). Thirty healthy adults 18 to 50 years of age received the III-TT vaccine adsorbed to alum. No increase in injection site pain, redness, or swelling was observed compared to III-TT conjugate without alum. Alum adsorption did not improve the immune response to III-TT conjugate (12.5-mg dose of III CPS). Four weeks after immunization, the GMC for recipients of the III-TT with and without alum were 3.3 and 3.6 mg/mL, respectively. Possibly because the human subjects were not immunologically naı¨ve to tetanus, as are mice or baboons, the TT protein carrier was not suitable for adjuvancy.

Maternal Immunization with GBS Type III Conjugate Vaccine Immunization during pregnancy is a strategy that takes advantage of the immunoresponsiveness of young healthy adults to provide protection passively for their infants. The value of this approach has been demonstrated globally with the eradication of tetanus neonatorum through widespread immunization of pregnant women with TT vaccine (44). Polysaccharide conjugate vaccines have also been administered safely to women during pregnancy. For example, Hemophilus influenzae type b conjugate vaccines have been administered safely to pregnant women in the United States and in developing countries (45). Two animal models were used to gain a better understanding of placental transfer of GBS CPS-specific IgG after maternal immunization with III-TT. In a murine model of infection, female outbred CD-1 mice received two intraperitoneal injections of III-TT and subsequently were bred (46). In sera from dams, CPS-specific IgG1 accounted for 83.2% of the total IgG, with a mean level of 9.1  3.9 mg/mL. Each of the 27 pups born to immunized dams survived challenge with a potentially lethal challenge of III GBS. In contrast, none of the 32 pups born to dams given placebo survived the same challenge dose. Since nonhuman primates produce antibodies that are structurally and functionally quite similar to those produced by humans, III-TT vaccine was evaluated in a baboon model of infection. Among immunized pregnant animals the GMC of III CPS-specific IgG increased from 0.9 mg/mL to 7.5 mg/mL at delivery. Seven of nine baboons had
4-fold rises in III-specific IgG from the baseline level. The percentage of III CPS-specific IgG transferred to their offspring ranged from 26% to 185% of maternal values. The function of the maternal and neonatal antibodies was equivalent when assessed in an opsonophagocytosis and killing assay.

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A prospective, randomized, double-blind, placebocontrolled trial was conducted to evaluate the safety and immunogenicity of III-TT vaccine in pregnant women. Thirty women, 18 to 45 years of age, received a single dose either of III-TT conjugate (12.5 mg) or placebo (0.9% saline) at a mean of 31 weeks of gestation (47). The vaccine was well tolerated. Mild injection site pain occurred in 65% of vaccine and 10% of placebo recipients; no redness or swelling was observed. All deliveries occurred beyond 37 weeks of gestation, and each neonate was healthy and had a normal Denver II developmental examination at six months. The III CPS-specific IgG GMC in sera of the 20 women receiving III-TT conjugate was 0.18 mg before and 9.98 mg/mL four weeks later. The GMC remained stable at the time of delivery (mean 9.76 mg/mL). Placebo recipients had 0.06 mg/mL of III CPS-specific IgG before and 0.05 mg/mL in sera after ‘‘immunization.’’ Vaccine recipients also developed immune response to the carrier protein, TT. The III CPS-specific IgG GMC in cord sera from infants whose mothers received vaccine was 7.48 mg/mL, reflecting 77% maternal delivery-cord III CPS-specific IgG transmission. The predominant IgG subclass of III CPS-specific antibodies was IgG2, as has been reported for Haemophilus influenzae type b conjugate maternal immunization (45). Cord serum values remained elevated at one (3.74 mg/mL) and two months (2.16 mg/mL) of age. Infant sera also promoted opsonization, phagocytosis, and killing of type III GBS, with 1.4 and 1.5 log10 kill, respectively, at one and two months of age. These findings suggest that maternal immunization has the potential to prevent early as well as late-onset GBS disease in infancy.

FUTURE VACCINE DIRECTIONS AND ISSUES Formulation and Target Population Improved immunogenicity through conjugation technology has been established in principle, but questions remain regarding formulation of an effective GBS vaccine for use in the United States and elsewhere. Because five CPS types (Ia, Ib, II, III, and V) are responsible for the majority of disease in the United States and Europe, a vaccine targeting protection against invasive disease caused by these CPS types appears to be warranted. That a multivalent GBS vaccine could confer protection against the types included in the vaccine has been demonstrated in mice with a tetravalent CPS (Ia, Ib, II, and III) conjugate vaccine formulation (24). Overcoming the technical challenges inherent in large-scale production of such a conjugate vaccine is akin to issues encountered by the developers of the sevenvalent pneumococcal conjugate vaccine (48), and is a task best left to experienced manufacturers. Formulation of a vaccine is predicated on knowing the amount of specific antibody required to be protective. This is not known with certainty for GBS, and is complicated in the case of neonatal disease by the necessity to induce predominately IgG class antibodies in the pregnant women to assure passive protection to the infant. However, the clinical studies summarized in the foregoing discussion suggest potential doses for each GBS type. Clearly, the maternal levels of CPS-specific antibodies in maternal delivery sera must readily exceed the concentrations presumed to confer protection in the newborn, which from earlier studies are in the range of 1 to 2 mg/mL (49). The amount of type-specific IgG elicited by GBS conjugate vaccines generally was dose-dependent. For type Ia, Ib, and III-TT conjugates there was no statistical difference in the CPS-specific IgG elicited between 50 to 60 mg and 12 mg to 15 mg

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doses. This latter dose rage is likely suitable for optimal immunogenicity. Type II-TT and V-TT conjugates elicited IgG responses at the lowest doses tested (3.6 mg and 2.4 mg, respectively) that suggest their adequacy for inclusion in a multivalent formulation (38,39). The formulation of a GBS conjugate vaccine should reflect the prevalence of GBS types causing invasive disease in the country of use. GBS colonization among pregnant women in Japan has been dominated by serotypes VI and VIII, which together accounted for 60% (44 of 73) of isolates (50). Although no clinical trials have been performed with type VI and VIII conjugate vaccines, preclinical studies in mice with VI-TT and VIII-TT vaccines suggest that effective vaccines against these serotypes can be achieved (51).

GBS Protein–Based Vaccines GBS possesses a number of surface-expressed proteins that have been tested as vaccine candidates or as carrier proteins for the CPS. Even if GBS proteins cannot, by themselves, provide protection against GBS disease, a multivalent GBS CPS conjugate would benefit from using protein carriers of GBS origin rather than TT and CRM197 that are in widespread use in licensed vaccines. A review of GBS surface components with potential for use in vaccines has been published (52). While all GBS protein antigens have shown promise in animal models of GBS disease, correlates between low antibody to GBS proteins and disease susceptibility in humans is lacking. a C Protein a C protein is expressed on *50% of clinical GBS isolates and on 70% of non-type III strains, so this laddering protein is an attractive candidate for inclusion in a GBS vaccine (53). Invasive human disease, but not colonization with a C-expressing GBS, elicits a C-specific IgM and IgG (54). Type III CPS conjugate vaccine prepared with a two-repeating unit a C protein conferred protection against GBS type III challenge in 95% of neonatal mice born to dams vaccinated before pregnancy, and 60% survival among pups challenged with a type Ia GBS strain containing a C protein (53). Most (73%) of the pups born to dams vaccinated with two-repeat a C protein survived lethal challenge with type Ia organisms, thus demonstrating the potential utility of this protein as a carrier for GBS CPS and an immunogen against a C protein-bearing GBS. b C Protein The b C protein is a 130-kDa trypsin-sensitive surface protein usually expressed by type Ib strains and some Ia, II, and IV GBS. In adults, bacteremia with b C-expressing strains of GBS elicits substantial concentrations of b C-specific IgM and IgG (55). In animals, GBS b C protein induces protective antibodies (56–58). A III CPS-b C protein conjugate elicited protective antibodies in mice against GBS strains bearing the type III antigen and b C protein (59). As b C protein also binds human IgA, efficacy of a recombinant GBS b C protein modified to eliminate its IgA binding site was tested in mice. Deletion of the IgA binding site did not alter antigenic b C protein epitopes and both the altered b C protein and III CPS-altered b C protein were immunogenic and protective as mouse maternal vaccines (60). C5a Peptidase Streptococcal C5a peptidase (SCPB) is a conserved surface protein among most GBS strains (61). Preincubation of GBS types Ia, Ib, II, III, and V with antibody to SCPB enhanced in

vitro opsonic-mediated killing by mouse bone marrow-derived macrophages (62). Moreover, GBS type III CPS covalently coupled to SCPB by reductive amination induced high levels of both CPS-specific and SCPB-specific IgG that opsonized not only GBS type III, but types Ib and V as well (62), suggesting cross-serotype activity. Immunization with recombinant SCPB alone or SCPB conjugated to type III CPS produced serotypeindependent protection that enhanced clearance of GBS from lungs (63). Rib Protein Found on GBS type III strains, Rib protein is structurally similar to but antigenically distinct from a C protein. Both proteins exhibit a laddering phenotype and are resistant to trypsin digestion (64). A low concentration of antibody to a and Rib in neonatal sera has been associated with GBS infection caused by Rib-expressing strains (65). Antiserum to this protein protected mice against GBS challenge, but only from GBS strains that did not contain a C protein. A bivalent vaccine composed of Rib and a C proteins was also protective (66). Sip Protein Surface immunogenic protein (Sip) is a highly conserved protein that induces cross-serotype immunity in mice (67). Using monoclonal antibody to its 53-kDa protein, Sip was present on representative strains of all nine GBS CPS types (68). Moreover, mice immunized with recombinant Sip were protected against infection with GBS types Ia, Ib, II, III, V, and VI, and newborn mouse pups born to Sip-vaccinated dams were effectively protected against challenge with types Ia, Ib, II, III, and V (69). These data suggest that cross-protective IgG to GBS can be induced by vaccination with a single surface protein common to all GBS types. Naturally occurring Sip antibodies are detectable in most (99%) pregnant women, and placental transfer to their newborn infants has been demonstrated, but obviously the amount of these detectable levels are not protective against infant disease. However, it is possible that Sip could be a GBS vaccine candidate (70). Leucine-Rich Repeat Surface Protein A gene, designated LrrG, that encodes a novel leucine-rich repeat surface protein that is conserved on strains of all nine GBS CPS types tested has been described (71). Recombinant LrrG protein adhered to epithelial cells, suggesting it may function as an adhesion factor. Immunization with LrrG elicited antibodies in mice that protected against lethal challenge, suggesting its promise as a candidate vaccine antigen. Pilus-like Structures Genome analysis has revealed that GBS produces long piluslike structures that extend from the bacterial surface (72). Formed by proteins with adhesive functions, these structures are implicated in host colonization, attachment, and invasion (73). The pilus-like structures are encoded in genomic islands with an organization that is similar to pathogenicity islands. Three types of pili that are composed of partially homologous proteins have been identified through genome analysis of multiple pathogenic isolates of GBS, and at least one of these is present on all GBS isolates tested (74,75). Recombinant pilus proteins protect mice from lethal challenge with GBS and, recently, an entire pilin island has been transferred from GBS to a nonpathogenic species where mucosally delivered Lactococcus expressing pilin island 1 protected mice from challenge

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with pilin 1-containing GBS strains (76). These findings, taken together, suggest that a pilus-based multivalent vaccine could provide broad protection against GBS.

SUMMARY Invasive GBS infection is a major health problem for infants, pregnant women, and nonpregnant adults. The emergence of type V strains as a cause of invasive disease, especially in nonpregnant adults and the elderly, highlights the need for ongoing epidemiological surveillance to correctly formulate a multivalent conjugate vaccine. The paucity of CPS types that contribute to human GBS disease by comparison, for example, with those documented to cause invasive pneumococcal infection, should be an advantage for the development of GBS conjugate vaccines. In an active statewide surveillance, Harrison and colleagues (77) characterized invasive GBS isolates from over 500 infants, children, pregnant women, and adults, and demonstrated that a pentavalent GBS vaccine, containing CPS from types Ia, Ib, II, III, and V GBS would theoretically provide protection against more than 95% of isolates from both infants and adults. The phase 1 and 2 trials of monovalent GBS CPS-protein conjugate vaccines conducted to date indicate the feasibility of developing a pentavalent GBS conjugate vaccine. The method for coupling to monomeric TT by reductive amination has generated custom vaccines of optimal immunogenicity. Each monovalent CPS-TT conjugate has been found safe, with only mild local reactions and, rarely, mild and time-limited systemic systems of less frequency than those associated with TT booster immunizations. These monovalent conjugates are highly immunogenic in healthy adults, with a more than fourfold increase in CPS-specific IgG from baseline in 80% to 100% of subjects. The response to immunization is durable, persisting for at least two years. The dose-dependence in the amount of CPS-specific IgG elicited indicates that the total CPS dose in a pentavalent vaccine is unlikely to exceed 50 to 75 mg. The recognition that adults 65 years of age and older constitute the population with the greatest overall mortality caused by GBS may provide an opportunity to document efficacy first in this population since pregnancy with its attendant liability issues loom strong after a decade of outcry for a perinatal disease prevention strategy more practical than IAP. Another approach that would avoid perceived liability associated with development of a maternal GBS vaccine would be immunological priming in girls aged 11 to 12 years when other adolescent vaccines are administered followed by a second dose when conception is anticipated. Ultimately, the licensure and widespread use of a multivalent GBS conjugate vaccine could result in thousands of lives saved and disabilities avoided annually in the United States alone.

ACKNOWLEDGMENTS Supported in part by Contracts #N01 AI-75236 and AI-25495 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.

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www.who.int/vaccines-diseases/diseases/NeonatalTetanus.htm. Accessed November 3, 2000. Englund JA, Glezen WP, Turner C, et al. Transplacental antibody transfer following maternal immunization with polysaccharide and conjugate Haemophilus influenzae type b vaccines. J Infect Dis 1995; 171:99–105. Paoletti LC, Pinel J, Kennedy RC, et al. Maternal antibody transfer in baboons and mice vaccinated with a group B streptococcal polysaccharide conjugate. J Infect Dis 2000; 181:653–658. Baker CJ, Rench MA, McInnes P. Immunization of pregnant women with group B streptococcal type III capsular polysaccharide-tetanus toxoid conjugate vaccine. Vaccine 2003; 21:3468–3472. Rennels MB, Edwards KM, Keyserling HL, et al. Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in United States infants. Pediatrics 1998; 101:604–611. Baker CJ, Edwards MS, Kasper DL. Role of antibody to native type III polysaccharide of group B Streptococcus in infant infection. Pediatrics 1981; 68:544–549. Lachenauer CS, Kasper DL, Shimada J, et al. Serotypes VI and VIII predominate among group B streptococci isolated from pregnant Japanese women. J Infect Dis 1999; 179:1030–1033. Paoletti LC, Pinel J, Johnson KD, et al. Synthesis and preclinical evaluation of glycoconjugate vaccines against group B Streptococcus types VI and VIII. J Infect Dis 1999; 180:892–895. Paoletti LC, Madoff LC, Kasper DL. Surface structures of group B Streptococcus important in human immunity. In: Fischetti VA, Novick RP, Ferretti JJ, et al. eds. Gram-Positive Pathogens. Washington, D.C.: ASM Press, 2000:137–153. Gravekamp C, Kasper DL, Paoletti LC, et al. Alpha C protein as a carrier for type III capsular polysaccharide and as a protective protein in group B streptococcal vaccines. Infect Immun 1999; 67:2491–2496. Pannaraj PS, Kelly JK, Rench MA, et al. Alpha C protein-specific immunity in humans with group B streptococcal colonization and invasive disease. Vaccine 2008; 26:502–508. Pannaraj PS, Kelly JK, Madoff LC, et al. Group B Streptococcus bacteremia elicits b C protein-specific IgM and IgG in humans. J Infect Dis 2007; 195:353–356. Fusco PC, Perry JW, Liang SM, et al. Bactericidal activity elicited by the beta C protein of group B streptococci contrasted with capsular polysaccharides. XIIIth Lancefield International Symposium on Streptococci and Streptococcal Diseases 1996, 201. Madoff LC, Michel JL, Gong EW, et al. Protection of neonatal mice from group B streptococcal infection by maternal immunization with beta C protein. Infect Immun 1992; 60:4989–4994. Michel JL, Madoff LC, Kling DE, et al. Cloned alpha and beta Cprotein antigens of group B streptococci elicit protective immunity. Infect Immun 1991; 59:2023–2038. Madoff LC, Paoletti LC, Tai JY, et al. Maternal immunization of mice with group B streptococcal type III polysaccharide-beta C protein conjugate elicits protective antibody to multiple serotypes. J Clin Invest 1994; 94:286–292. Yang H-H, Madoff LC, Guttormsen H-K, et al. Recombinant group B Streptococcus beta-C protein and a variant with the deletion of its immunoglobulin A-binding site are protective mouse maternal vaccines and effective carriers in conjugate vaccines. Infect Immun 2007; 75:3455–3461. Suvorov AN, Cleary PP, Ferrieri P. C5a peptidase gene from group B streptococci. In: Dunny GM, Cleary PP, McKay LL, eds. Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci. Washington, D.C.: American Society for Microbiology, 1991:230–232. Cheng Q, Carlson B, Pillai S, et al. Antibody against surface-bound C5a peptidase is opsonic and initiates macrophage killing of group B streptococci. Infect Immun 2001; 69:2302–2308. Cheng Q, Debol S, Lam H, et al. Immunization with C5a peptidase or peptidase-type III polysaccharide conjugate vaccines enhances clearance of group B streptococci from lungs of infected mice. Infect Immun 2002; 70:6409–6415.

Chapter 63: Vaccines Against Group B Streptococcus 64. Stalha˚mmar-Carlemalm M, Stenberg L, Lindahl G. Protein Rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections. J Exp Med 1993; 177:1593–1603. 65. Larsson C, Lindroth M, Sta˚lhammar-Carlemalm M, et al. Association between low concentrations of antibodies to protein a and Rib and invasive neonatal group B streptococcal infection. Arch Dis Child Fetal Neonatal Ed 2006; 91:F403–F408. 66. Larsson C, Sta˚lhammar-Carlemalm M, Lindahl G. Protection against experimental infection with group B streptococcus by immunization with a bivalent protein vaccine. Vaccine 1999; 17:454–458. 67. Rioux S, Martin D, Ackermann HW, et al. Localization of surface immunogenic protein on group B Streptococcus. Infect Immun 2001; 69:5162–5165. 68. Brodeur BR, Boyer M, Charlebois I, et al. Identification of group B streptococcal Sip protein, which elicits cross-protective immunity. Infect Immun 2000; 68:5610–5618. 69. Martin D, Rioux S, Gagnon E, et al. Protection from group B streptococcal infection in neonatal mice by maternal immunization with recombinant Sip protein. Infect Immun 2002; 70: 4897–4901. 70. Manning SD, Wood S, Kasha K, et al. Naturally occurring antibodies for the group B streptococcal surface immunogenic protein

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64 Overview of Live and Subcellular Vaccine Strategies Against Shigella Karen L. Kotloff, Eileen M. Barry, and Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

Thomas L. Hale Department of Enteric Infections, Division of Communicable Disease and Immunology, Walter Reed Army Medical Center, Washington, D.C., U.S.A.

Philippe Sansonetti Unite´ de Pathoge´nie Microbienne Mole´culaire, Institut Pasteur, Paris, France

SIGNIFICANCE AND BURDEN OF SHIGELLA INFECTION Clinical and Epidemiological Features Shigella is a pathogen found globally that is capable of rapid dissemination in settings where there is overcrowding, inadequate sanitation, and insufficient supply of clean water or when personal hygiene is compromised. The spectrum of symptoms ranges from mild watery diarrhea to fulminate bacillary dysentery, characterized by bloody stools, high fever, prostration, cramps, and tenesmus. An array of severe intestinal and extraintestinal complications can occur, such as hemolytic-uremic syndrome and a protein-losing enteropathy, which contribute disproportionately to diarrhea-related malnutrition and mortality in young children. Most of the disease burden from shigellosis result from endemic disease among children of one to four years living in developing countries (1). However, one serotype, Shigella dysenteriae type 1, produces devastating epidemics and pandemics with high case fatality rates in all age groups, often appearing in populations experiencing political upheaval and natural disaster (2). In industrialized countries, Shigella causes a small proportion of endemic pediatric diarrhea and also occurs among persons who belong to known risk groups, such as individuals residing in institutions and soldiers deployed to endemic regions. High infectivity and antimicrobial resistance are factors that have enabled this bacterium to elude routine control measures. Development of a safe and effective Shigella vaccine has long been considered a desirable strategy for combating this infection.

Shigella Serotypes of Clinical and Epidemiological Importance Shigella is an antigenically diverse pathogen representing four species (or groups) and 50 serotypes and subserotypes. Among the 15 serotypes belonging to S. dysenteriae (group A), type 1 poses the greatest threat because of its ability to produce

pandemics (3–5). The other 14 S. dysenteriae serotypes are uncommon diarrheal pathogens and lack the factors that confer excessive virulence to type 1, such as production of Shiga toxin (Stx). S. flexneri (group B) comprises 14 classical serotypes and subserotypes, defined by type (I, II, IV–VI) and group (3,4; 6; 7,8) antigens, which together represent the major cause of endemic shigellosis among children in developing countries (1). The 20 serotypes of S. boydii (group C) appear with relative infrequency. On the other hand, S. sonnei (group D), which has a single serotype, is the major cause of shigellosis in industrialized and transitional countries.

IMMUNITY TO SHIGELLA AS IT RELATES TO VACCINE DEVELOPMENT Evidence of Protective Immune Responses Demonstration, in several venues, that an initial wild-type Shigella infection prevents illness following subsequent exposure provides a compelling argument for using a live, oral, attenuated vaccine approach (6–8). The protection seen in efficacy field trials with early live oral noninvasive Shigella vaccines offers additional hope that a live vaccine can succeed (9,10). To date, approaches that have conferred clinical protection have not prevented intestinal colonization (9,10), although fecal excretion has been reduced (8).

Homotypic vs. Heterotypic Immunity Serotype-specific immunity has been shown unequivocally in field (6,9,10) and clinical (7,8) settings; however, the existence of heterotypic immunity is less certain. Cross-protection has been induced in animal models among S. flexneri serotypes. Noriega et al. observed that two serotypes, S. flexneri 2a and 3a, share a type or group-specific antigen on the O-polysaccharide molecule with all other S. flexneri serotypes except for type 6, suggesting that a vaccine containing serotypes 2a, 3a, and 6

Chapter 64: Live and Subcellular Vaccine Strategies Against Shigella

could provide protection against the remaining 12 S. flexneri (11). This hypothesis translated into significant cross-protection when guinea pigs that received a S. flexneri 2a/3a combination vaccine were challenged with wild-type strains 1b, 2b, 4b, and 5b, which share group and/or type antigens (11). It also explains why a polyvalent vaccine containing attenuated strains of S. flexneri 1b, 2a, and 3 and S. sonnei protected monkeys against challenge with the homologous wild-type parent but not against challenge with S. flexneri 6 (12). Moreover, a S. flexneri Y strain protected monkeys against challenge with S. flexneri 2a, with which it shares group antigen ‘‘3,4’’ (13). It also protected against S. flexneri 1b, which does not share known antigens, suggesting there may be other common antigenic sites among S. flexneri serotypes (13). There is also evidence for heterologous serotype protection in humans. Sera from subjects who had previously received a S. flexneri 2a vaccine or wild-type challenge strain cross-reacted with serotype 2b (which shares a type II antigen), and serotypes 1a, 5a, and Y (and, to a lesser extent, 3b and 4a), which share group 3,4 antigens with 2a (14). Immunoglobulin A (IgA) was the predominant cross-reacting antibody. A field trial evaluating the efficacy of streptomycindependent (SmD) S. flexneri 2a vaccine concluded that protection was limited to the homologous serotype, suggesting that experimental cross-reactivity did not translate into clinical immunity (9). However, the attack rate of S. flexneri serotypes not in the vaccines was too low to assess heterologous immunity definitively. Although wild-type infection elicits immune responses to Shigella plasmid-encoded proteins (e.g., IpaA-D and VirG/ IcsA) that are shared among the four serogroups (15,16), the existence of cross-species protection is unproven. Monkeys fed wild-type S. flexneri 2a were fully protected when rechallenged with the same strain, but all monkeys became ill upon challenge with S. sonnei (17). The SmD Shigella vaccine field trials did not accrue sufficient numbers of unvaccinated control subjects to assess cross-species protection (9,10,18). In contrast, investigators conducting field trials in Romania (19) and in China (20) in the 1970s to 1980s claimed that the T32-Istrati S. flexneri 2a vaccine provided both cross-species and crossserotype protection. Additional investigation into this important area is needed to guide vaccine development.

Specific Immunoprotective Responses The above examples of serotype-specific protection support the hypothesis that immunity is mediated by antibodies (serum or mucosal) directed against the lipopolysaccharide (LPS) O-antigen (21,22). The efficacy of parenteral S. sonnei O-specific polysaccharide conjugate vaccine in preventing S. sonnei disease among Israeli soldiers (although an immunologically primed population) lends further credence to this concept (23). The first line of defense must occur at the mucosa. The impact of mucosal immunity is illustrated by the efficacy of passively transferred type-specific oral immunoglobulin (Ig) in preventing shigellosis (24) and perhaps the ameliorating effects of breast-feeding on disease severity in developing countries (25). In healthy adult volunteers, gut-derived O-specific IgA antibody-secreting cells (ASCs) circulating in the bloodstream 7 to 10 days after oral vaccination is a measure of intestinal priming that has been correlated with vaccine efficacy (26,27).

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Cell-mediated immune responses may also contribute to the defense against this intracellular pathogen. In animal models, interferon gamma (IFN-g) production by natural killer (NK) cells seems to be an essential component of innate immunity to Shigella infection (28). Upregulation of IFN-g production and expression of IFN-g receptor are seen in the epithelium of rectal biopsies from Bangladeshi patients convalescing from shigellosis (29), and elevated levels are found in both serum and stool (30). IFN-g is the predominant cytokine produced by T cells in response to Shigella antigens, including highly purified Ipa proteins, following inoculation of volunteers with liveattenuated Shigella vaccine candidates (31–33). Nonetheless, prospective studies have not yet been performed in humans to correlate cell-mediated immune responses with protection.

PATHOGENESIS Rupture, Invasion, and Inflammatory Destruction of the Intestinal Epithelium by Shigella: An Emerging Scheme Genetic, Molecular, and Cellular Bases of the Invasive Phenotype: The Virulence Plasmid of Shigella The invasive phenotype that characterizes Shigella pathogenesis encompasses several steps. First, the bacteria traverse the follicle-associated epithelium (FAE) located over gut mucosal lymphoid nodules, the inductive sites of the local immune response (34). Next, they are engulfed by phagocytic cells located in the dome of the FAE (29). The organisms invade the epithelial lining of the intestine through its basolateral pole by a macropinocytic event made possible by reorganization of the host cell cytoskeleton. Finally, the Shigella bacteria spread cell to cell. A 214-kb virulence plasmid carries most of the genes required for invasiveness (35), including a 30-kb pathogenicity island, which encodes a type III secretion system (TTSS, mxi-spa operons) and at least five effector proteins (IpaA-D, IpgD) that are secreted through this TTSS (36). IpaB and IpaC form a pore in the cytoplasmic membrane of the target cell through which the bacterium injects its effector proteins. IpaC induces a signaling cascade involving small Rho GTPases Rac1 and Cdc42, which recruit the actin-nucleating complex Arp2/3 (37), forming localized filopodes and lamellipodes that entrap the bacterium. After cell entry and lysis of the phagocytic vacuole, Shigella enters the cytoplasm, replicates, and moves intracellularly by a process mediated by the outer membrane protein, VirG/IcsA (38,39). VirG/IcsA localizes to one pole of the bacterium and engages host cell N-WASP, forming a complex with Arp2/3, which elicits actin polymerization/ nucleation; elongation of the actin tail results in bacterial motility (40). Shigella then engages with the intermediate junctions and propels itself via protrusions that are engulfed by adjacent cells (41). Figure 1 summarizes this remarkable mechanism of epithelial colonization that is contained within the sanctuary of the intracellular environment. Inflammatory Destruction of the Intestinal Epithelium Shigella produces inflammation and destruction of the intestinal epithelium by several mechanisms that are illustrated in Figure 1. For one, macrophages that internalize Shigella in the FAE undergo apoptotic death through a process that involves activation of Caspase-1 by IpaB, resulting in release of proinflammatory cytokines such as interleukin (IL)-1b and IL-18 (42). The epithelial cell response to intracellular Shigella includes secretion of IL-8

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Figure 1 Rupture, invasion, and inflammatory destruction of the intestinal epithelium by Shigella: an emerging scheme. Abbreviations: PMN, polymorphonuclear leukocyte; TTSS, type III secretion system.

(43) and rapid, sustained activation of the proinflammatory cytokine transcription system nuclear factor kappa B (NF-kB) through a signaling cascade that involves cytosolic proteins of the Nod family, particularly Nod1 (40). Inflammation, in turn, enhances invasion by disrupting the epithelial barrier and facilitating access of the bacteria to sites that permit epithelial invasion. Eventually this process leads to massive chemotaxis of neutrophils and bacterial killing, although at the cost of epithelial destruction (44). Endotoxin (i.e., lipid A of Shigella) is necessary for expression of the invasive phenotype and also contributes to the rupture and destruction of the epithelial lining, mostly through tumor necrosis factor alpha (TNF-a) (43). Nonetheless, Shigella is able to establish infection and direct the nature of adaptive immunity subsequently induced by commandeering the innate immune response (45). Numerous plasmid-encoded proteins that are secreted by the TTSS contribute to this process (46). Shigella co-opts the ubiquitination pathway either to promote [e.g., via IpaH (47)] or prevent [e.g., via OspG (48)] degradation of host proteins to downregulate host inflammatory responses. OspF alters transcription of a subset of NF-kB genes, particularly il-8, thus inhibiting neutrophil transmigration through the intestinal epithelium (49). OspF also regulates the adaptive immune response to Shigella by suppressing the IL-12, IFN-g [T helper 1 (Th1)] type response (Gamelas et al., submitted), creating an immunosuppressive state that may impair the immunogenicity of candidate vaccines. These data, overall, may need to be taken into account for the development of the next-generation of live attenuated Shigella vaccines.

Role of Toxin in Shigella Pathogenesis Shigella elaborates several toxins that contribute to virulence. S. dysenteriae type 1 is virtually the only serotype that produces Stx, a potent cytotoxin that cleaves eukaryotic ribosomal RNA, thus inhibiting protein synthesis and inducing cell death. The pathogenesis of hemolytic uremic syndrome has been attributed to the ischemic and prothrombotic effects of circulating Stx

that binds to the microvascular endothelial cells of renal glomeruli and possibly other target cells. Two enterotoxins have been identified in rabbit ileal loop and Ussing chamber studies as likely mediators of the watery diarrhea often seen in early shigellosis (50–52) or as a component of the reactogenicity that has beset live oral vaccine candidates (27,32,53). Shigella enterotoxin (ShET) 1 is an A1-B5 subunit protein (52) encoded by setBA on a SHI-1 chromosomal pathogenicity island and found almost exclusively in S. flexneri 2a (50). The setBA operon is completely embedded within a larger gene on the opposite DNA strand that encodes Pic (originally termed ‘‘ShMU’’) (54), a serine protease autotransporter that may participate in colonization by Shigella (55). ShET2 is a single-moiety protein encoded by sen on the invasion plasmid of nearly all Shigella serotypes (51). Watery diarrhea could also result from release of inflammatory mediators by colonic leukocytes (56) or by upregulation of receptors such as galanin-1 in inflamed epithelium (57).

Rational Construction of Live-Attenuated Shigella Strains Guided by Molecular Pathogenesis The evolving understanding of the molecular pathogenesis of shigellosis guides the design of rational live-attenuated vaccines. For example, the adeptness of invasive strains to traverse the FAE to reach the inductive sites of the mucosal immune system has been exploited to enhance the immunogenicity of vaccine candidates. Recently, an alternative approach has been explored whereby Shigella’s invasive phenotype is eliminated by creating a DipaB mutation, while the strain is provided with potential adhesive capacity to M cells by expressing the Yersinia Inv molecule (58). The benefits of retaining triggers of macrophage apoptosis remain uncertain. On the one hand, this process generates a strong proinflammatory signal (59) that must be mitigated; on the other hand, apoptotic bodies of infected antigen-presenting cells are strongly immunogenic. Eliminating cell-to-cell spread improves vaccine tolerance by minimizing the extent of the destructive intestinal lesion. The propensity for Shigella to cause watery diarrhea can be

Chapter 64: Live and Subcellular Vaccine Strategies Against Shigella

diminished with mutations in genes encoding enterotoxin synthesis. Clearly any S. dysenteriae 1 vaccine should be devoid of catalytically active Stx and harbor additional attenuating mutations because S. dysenteriae 1 strains not expressing Stx retain their pathogenicity in humans (60). Because of the propensity for S. dysenteriae 1 to appear in disrupted and displaced populations, a desirable feature of a vaccine is its amenability to use in emergency situations. As knowledge of the molecular mechanisms of the innate immune response unfolds, it may become possible to suppress proinflammatory pathways while stimulating desired adaptive immune responses.

MODELS FOR EVALUATION OF VACCINE CANDIDATES Animal Models The rhesus monkey model is often used to assess the efficacy of live oral vaccines. The histological response to intragastric inoculation closely mimics that seen during human infection, and diarrhea and dysentery are the principal clinical manifestations (61). Small animal models have been developed for their practicality and lower cost. The Sereny test is a well-established model that measures the ability of Shigella inoculated into the conjunctival sac of animals (usually guinea pigs) to cause purulent keratoconjunctivitis (62). Accepted practice in Investigative New Drug applications is to show that a live Shigella vaccine is Sereny test negative before initiating phase I trials. Nonetheless, some vaccines that are fully attenuated in the Sereny model can be reactogenic in humans at the high end of dose-ranging studies. The model also has been adapted to assess vaccine efficacy (63). An alternative guinea pig model has recently been proposed in which animals inoculated intrarectally with invasive S. flexneri develop an acute rectocolitis that clinically and histopathologically mimics human bacillary dysentery (64). A live-attenuated S. flexneri 2a vaccine candidate (SC602) appeared attenuated and protective in this model as it did in humans. However, this new model will probably not be as sensitive as the Sereny test for demonstrations of vaccine safety. The mouse intranasal Shigella challenge model produces a pulmonary infection that is histologically similar to the colitis seen with human shigellosis (65). The availability of inbred mouse strains and cytokine reagents allows characterization of the immune response generated by Shigella vaccines on a molecular level, but this model, like the Sereny test, can only hint at the complexity of a protective immune response in the human colon.

Volunteer Models for Vaccine Efficacy Ultimately, the safety and efficacy of a Shigella vaccine must be determined by studies in humans. The modern experimental challenge model of Shigella in volunteers was developed in the 1960s (60) and its ability to detect protective efficacy has been validated using homologous strain rechallenge studies (7,8,66) that confirmed the immunizing ability of clinically overt infections with wild-type strains as observed in the field. Moreover, the S. flexneri 2a SmD vaccine that was efficacious in field trials (10) conferred significant protection in this model (Table 1) (66). In the 1990s, the S. flexneri 2a model was modified to administer the inoculum in sodium bicarbonate buffer rather than skim milk, which safely increased the attack rate and consistency of

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induced illness in control subjects and lowered the sample sizes needed for an individual trial (8).

LIVE SHIGELLA VACCINE STRAINS LACKING THE INVASIVE PHENOTYPE Streptomycin-Dependent Vaccines Mel et al. developed SmD mutants of S. sonnei and certain S. flexneri serotypes by serial passage on streptomycin-containing media (73). The basis of attenuation is now believed to be spontaneous deletion of virulence genes from the invasion plasmid (F. Noriega, unpublished). Monovalent and bivalent combinations of S. flexneri (serotypes 1, 2a, and 3) and S. sonnei were safe and protective (82–100% efficacy) when given (with buffer) to nearly 8000 adults and children in controlled field trials in Yugoslavia (10,18,72). Both freshly harvested and lyophilized formulations were used. Vaccine was excreted by 90% of recipients for a mean of three days (72). However, drawbacks of these vaccines are recognized, including (i) the need for multiple (4–5) doses, the large inocula (2–6
1010 CFU) required per dose (which affects cost) (9,10,18); (ii) the occurrence of vomiting and less often mild diarrhea (usually after the first dose), which was minimized by giving gradually increasing inocula (9,10,18); (iii) the need for a booster dose at one year to maintain protection (68); (iv) occasional in vivo reversion to streptomycin independence (without loss of attenuation) (74,75); and (v) equivocal efficacy in preventing endemic shigellosis among institutionalized children in the United States (76).

T32-Istrati Strain Romanian scientists used the Sereny test as a screening device to identify spontaneous avirulent mutants of S. flexneri 2a. After 32 passages on nutrient agar, a mutant was identified as consistently Sereny negative. This strain, designated T32Istrati, was tested as a live oral vaccine in Romania (77) and China (20) in the 1970s. Decades later, the genetic basis of attenuation was shown to be a consequence of a spontaneous 32-MD deletion in the invasion plasmid, which eliminated ipa and other virulence determinants such as virF (a positive regulator for ipa genes) and virG/icsA (78). The Cantacuzino Institute of Bucharest manufactured T32-Istrati under the trade name Vadizen as a liquid, refrigerated vaccine that was administered with buffer as 5 oral doses increasing from 5
1010 CFU to 2
1011 CFU at three-day intervals. Open-label Romanian trials involving 37,000 adults and children suggested 81% protection against S. flexneri 2a using historical data for comparison. These studies also reported 89% protection against heterologous Shigella species including S. sonnei (77). Later, placebo-controlled field studies in China involving 11,128 adults and children were conducted using an enteric-coated, refrigerated version of T32-Istrati (without buffer) manufactured by the Lanzhou Institute of Biological Products. In these trials, the T32 again appeared well tolerated. Most recipients excreted vaccine for two to four days after the last dose. Efficacy was reported as 85% against homologous disease and 63% against heterologous strains identified as S. flexneri 1b and S. boydii 1–6 (20).

FS Bivalent Vaccine S. sonnei is unique among Shigella species in that the rfb locus encoding the form I (smooth) O-polysaccharide of LPS is carried on a 120-MD invasion plasmid. In 1987, Wang et al. at the

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Table 1 Clinical and Immune Response of Adult Volunteers from Industrialized Countries to Wild-Type Infection and Live, Attenuated Shigella flexneri 2a and Shigella dysenteriae 1 Vaccine Candidates Anti-LPS responsesa

Immunogen

Parent strain

Reference

Shigella flexneri 2a Wild type

2457T

8

Streptomycindependent vaccines EcSf2a-2

2457T

66

na

26,61

SC602

454

27,67

SFL1070

2457T

68

CVD 1203

2457T

53

CVD 1207

2457T

69

CVD 1208S

2457T

33

Shigella dysenteriae 1 WRSd1e 1617 7/87 SC599f

70 71

Percentage with response

lgA antibody secreting cell responses Geometric Mean

Serum lgG antibody

Protective efficacy vs. challenge

239 18 nd

50 29 38

70 nd 49b

53c 19 35c 20 10 67% 22 46 45 30 17 17 0 0 14 43 0

48 27 0 nd 50 nd nd nd nd nd nd nd nd nd nd nd nd

Dose (CFU)

Percentage with adverse reactions

103 102 1010

92 43 0–15

109 108 109 106 104 109 108 109 108 106 1010 109 108 107 106 109 108

0 10 17 60 20 44 0 80 12 0 20 8 0 0 0 14 14

100 100 93 93 66 89 89 100 91 60 100 64 67 100 0 57 29

59 16 21 154 26d 10 5 175 43 13 35 9 5 6 0.1 38 12

103–107 105

20 32g 11h 14g 3h

60 34

6 8

12 10

nd nd

44

11

6

nd

107

92 71 nd

a Study-specific definitions of ASC responses were used. IgG antibody responses were defined as the fourfold rises, with the exception of the SC602 study which considered a threefold rise to be a response. b Protection in field trial was 82% to 100% (9,10,18,72). c The protective efficacy of these two trials combined was 36%. d The geometric mean was 43 among volunteers who participated in the challenge trial. e No dose response was seen so responses to all doses were combined. f Results of phase II study shown. g percentage with diarrhea. h percentage with fever. Abbreviations: GM, geometric mean expressed per 106 PBMC; na, not applicable; nd, not done; Ig, immunoglobulin, CVD, Center for Vaccine Development.

Lanzhou Institute of Biological Products mobilized a plasmid from a noninvasive strain of S. sonnei into T32-Istrati, thereby creating a bivalent S. flexneri–S. sonnei (FS) vaccine (79,80). Since this S. sonnei plasmid did not hybridize with probes for ipa or virF (81), it had probably suffered a spontaneous deletion of invasion loci similar to that seen in T32-Istrati. Two double-blind, placebo-controlled efficacy trials of the FS vaccine were conducted in China. A three-dose regimen using a lyophilized FS product was administered at 2–5
1010 CFU to 17,500 adults and children, while 15,700 subjects received placebo. Passive surveillance for shigellosis was conducted for five to six months. Protective efficacy was reported to be 61% to 65% against S. flexneri 2a disease, 50% to 72% against S. sonnei, and 48% to 52% against heterologous Shigella species (82). These data indicate that the noninvasive T32-Istrati and FS hybrid vaccines are clinically well tolerated, safe, and confer significant protection against both homologous and heterologous serotypes. The reported 60% protection against shigellosis

could have a positive public health impact. However, the immunological basis of heterologous protection remains unclear. In addition, the need for a three- to five-dose regimen containing over 10-logs of live bacteria is a daunting logistical prospect for most developing countries.

HYBRID VACCINES Bivalent Salmonella Typhi-Shigella Sonnei Hybrid Vaccine In the 1980s, efforts to develop improved live Shigella vaccines included heterologous carrier (i.e., live vector) strains designed to invade and proliferate within the intestine to deliver Shigella antigens more effectively to the local immune system. Strain 5076-1C utilized Ty21a, an invasive attenuated vaccine strain of Salmonella enterica serovar Typhi, to carry the form I S. sonnei plasmid expressing S. sonnei O-antigen. In volunteers, a lyophilized formulation of 5076-1C was well tolerated and in initial

Chapter 64: Live and Subcellular Vaccine Strategies Against Shigella

trials protected against experimental S. sonnei challenge (22). However, the efficacy results could not be replicated with subsequent lots of vaccine and further clinical development was not undertaken (7). Chemical and immunoblot analyses of 5076-1C indicated that the O-polysaccharide of S. sonnei was transported to the cell surface without covalent linkage to the core polysaccharide or the lipid A of S. Typhi, perhaps accounting for its erratic immunogenicity (83).

Escherichia Coli as a Carrier for Shigella Antigens In the 1970s, a noninvasive Escherichia coli carrier vaccine expressing S. flexneri 2a O-antigen was evaluated by Levine et al. (84). Although the vaccine was safe and colonized the intestinal tract of volunteers for several days, antibody responses to the O-antigen were meager and the vaccine did not confer protection against wild-type challenge. To enhance immunogenicity, second-generation E. coli carrier vaccine strains were constructed containing the invasion plasmid of S. flexneri 5 plus the chromosomal genes encoding S. flexneri 2a LPS (85). These strains were invasive and expressed O-antigen but were attenuated in the Sereny test (85). An initial construct, EcSf2a-1, produced adverse reactions in subjects who ingested 109 CFU, while better-tolerated doses (ca. 106–107 CFU) did not protect against challenge (61). A further attenuated DaroD mutant of EcSf2a-1 was constructed and designated EcSf2a-2 (86). EcSf2a-2 induced fever and/or diarrhea in 11% of subjects who received three doses of 109 CFU over a one-week span. Although most subjects had anti-LPS immune responses, the vaccine conferred only 36% protection against challenge (Table 1) (61). Subsequent studies attempted to improve safety by giving a lower inoculum (7
108 CFU) and to enhance efficacy using a four-dose regimen. Unfortunately, immunogenicity was modest and vaccine efficacy was only 27% (Table 1) (26).

ENTEROINVASIVE DELETION-MUTANTS OF SHIGELLA Molecular engineering has enabled the development of live, oral Shigella vaccine candidates with genetically defined mutations. The strains described below retain invasiveness but are attenuated by deletions that either induce auxotrophy for metabolites that are essential for survival of the bacteria in vivo, such as genes in the aromatic or purine metabolic pathways, and/or inactivate virulence factors, such as VirG/IcsA and the ShETs 1 and 2.

Aromatic Auxotrophs Auxotrophic S. flexneri Y Vaccine Candidates Mutations in genes involved in aromatic amino acid synthesis pathway prevent intracellular bacteria from making folic acid de novo (Fig. 2). If environmental sources are not available, the growth of the mutant is severely hindered (93). Lindberg et al. applied this strategy to construct Shigella vaccine strain SFL124 with a transposon-generated 1400 base pair deletion in aroD of virulent S. flexneri Y strain SFL1 (89). They postulated that a serotype Y strain might provide cross-protection against other S. flexneri serotypes since its O-polysaccharide shares tetrasaccharide repeating units with all other S. flexneri except serotype 6. A dose of 109 CFU was given with buffer to adult volunteers living in Sweden (94) and Vietnam (95), and to 9- to 14-year-old Vietnamese children (96). SFL124 appeared safe and elicited both primary and anamnestic immune responses.

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However, subsequent studies in North American volunteers showed that the wild-type S. flexneri Y parent strain SFL1 was only minimally diarrheagenic. Auxotrophic S. flexneri 2a Vaccine Candidates The Swedish investigators also evaluated a DaroD S. flexneri 2a strain 2457T called SFL1070 (97) in Swedish adults. SFL1070 exhibited a dose-dependent pattern of clinical effects, vaccine excretion, and immune responses (68). Transient mild gastrointestinal symptoms occurred in 10% to 33% of subjects who received three doses of 105 to 108 CFU within five days. Vaccination with 109 CFU caused more severe symptoms in 44% of volunteers (Table 1). These results point to the importance of the inherent virulence of the parent strain in dictating the clinical tolerance of vaccines with identical attenuating lesions. The parent strain of SFL1070 is more pathogenic in humans than the SFL1 strain used to construct SFL124. Auxotrophic S. flexneri 2a with a Deletion in virG Noriega et al. attempted to enhance the attenuation of S. flexneri 2a 2457T conferred by aromatic auxotrophy by introducing a second, specific, in-frame, deletion in the plasmid gene icsA/ virG, resulting in double mutant DaroA,DicsA/virG S. flexneri 2a strain CVD 1203 (98). These specific deletions were created by allelic exchange of the modified genes for their wild-type counterparts using suicide vectors (98). A phase I study was conducted at the Center for Vaccine Development (53). After a single dose of ca. 109, 108, or 106 CFU with buffer (10–11 subjects/group), brief clinical reactions (fever, diarrhea, and/ or dysentery) occurred in 72%, 18%, and 0% of volunteers, respectively. Anti-LPS IgA ASC responses were dose related, occurring even at doses of 106 CFU where illness was not seen (Table 1). A striking component of the reactogenicity observed with CVD 1203 was the occurrence of watery diarrhea in approximately 25% of those who receive 108 or 109 CFU (94), suggesting that deletion of genes involved in production of ShETs may further attenuate Shigella vaccines.

Purine Auxotrophs Stimulated by the observations of McFarland and Stocker on the attenuating effects of purine auxotrophy in the mouse model of Salmonella virulence (87), Noriega et al. constructed a lineage of Shigella vaccine candidates from S. flexneri 2a strain 2457T based on a mutation in the genes encoding two enzymes in the guanine nucleotide biosynthetic pathway, guanosine 5’-monophosphate (GMP) synthetase (guaA) and inosine 5’monophosphate dehydrogenase (guaB) (99) (Fig. 1). Using homologous recombinations with a suicide plasmid bearing the deleted alleles, they guanosine first created the DguaBA strain CVD 1204 (88). A series of strains with additional mutations was made in search of the optimal balance of safety and immunogenicity. To create CVD 1207 (DguaBA, DvirG,Dsen, Dset), a second in-frame deletion was made in the plasmid gene virG (88). The chromosomal mutation Dset was accomplished with deletion of 85% of the subunit A (11). Dsen was constructed by fusing the N and C termini of sen minus 300 bp corresponding to the putative active site in the N-terminal region (11). An ars operon, conferring resistance to arsenite, was cloned into the Dsen to facilitate transfer of the mutated virulence plasmid to other Shigella vaccine strains and as a marker to distinguish CVD 1207 in the field (69). Finally,

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Figure 2 Purine de novo biosynthesis pathway and contribution of the aromatic metabolic pathway. Interrupted arrows illustrate pathways in which the individual steps are not represented. Enzymes are represented by their genes. Superscript letters represent selected published strains with a mutation of the gene involved in that reaction. Strains represented are (a): purF1741::Tn10 S. dublin SL5437 (87); (b): purG876::Tn10 S. dublin SL5436 (87); (c): purC882::Tn10 S. dublin SL5435 (87); (d): purH887::Tn10 S. dublin SL2975 (87); (e): DguaBA S. flexneri 2a CVD1204 and DguaBA, DvirG CVD1205 (88); (f): aroD25::Tn10 S. flexneri Y SFL114 (89), SFL124 (90), S. flexneri 2a 1070 (88); (g): DaroC,DaroD S. typhi CVD908 (91); (h): hisG46 DEL407,aroA554::Tn10 S. typhimurium SL3261 (82); (i): DaroA S. flexneri 2a CVD1201 and DaroA,DvirG CVD1203 (89); (j): DaroA,DhisG,DpurA S. typhi 541Ty (92). Abbreviations: PRPP, 5-phosphoribosyl-1-pyrophosphate; PRA, 5-phosphoribosylamine; GAR, 50 -phosphoribosyl-1-glycinamide; FGAR, 50 -phosphoribosyl-N-formylglycinamide; FGAM, 50 -phosphoribosyl-Nformylglycinamidine; AIR, 50 -phosphoribosyl-5-aminoimidazole; CAIR, 50 -phosphoribosyl-5-aminoimidazole-4-carboxylic acid; SAICAR, 50 -phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole; AICAR, 50 -phosphoribosyl-4-carboxamide-5-aminoimidazole; FAICAR, 50 -phosphoribosyl-4-carboxamide-5-formamidoimidazole; IMP, inosinic acid; Hx, hypoxanthine; G, guanine; A, adenine.

Chapter 64: Live and Subcellular Vaccine Strategies Against Shigella

DguaBA,Dsen,Dset strain CVD 1208 was derived by restoring wild-type virG. The lack of enterotoxic activity of CVD 1207 and 1208 was confirmed in Ussing chambers (11). Phase I Trial of CVD 1207 (DguaBA,DvirG,Dsen::ars,Dset S. flexneri 2a) Investigators at the Center for Vaccine Development evaluated CVD 1207 for safety and immunogenicity. Incremental doses ranging from 106 to 1010 CFU were administered to volunteers (Table 1) (69). Vaccination was well tolerated at doses as high as 108 CFU. In comparison, 1 of 12 recipients of 109 CFU and 1 of 5 recipients of 1010 CFU experienced mild diarrhea and emesis. All recipients of 108 to 1010 CFU excreted vaccine, most for fewer than three days, although two subjects had positive stool cultures 14 days postvaccination and were treated with ciprofloxacin. Geometric mean peak IgA ASC anti-LPS responses of 6 to 35 ASCs per 106 peripheral blood mononuclear cell (PBMC) were seen among recipients of 107 to 1010 CFU. The array of mutations in CVD 1207 thus achieved a remarkable degree of attenuation of virulent Shigella compared with experiences with earlier invasive S. flexneri 2a recombinant strains. However, occasional adverse reactions occurred at high inocula, and at well-tolerated doses, CVD 1207 appeared insufficiently immunogenic after a single dose. Phase I Trials of CVD 1204 (DguaBA) and the Attenuating Effects of Adding Dsen and Dset to Create CVD 1208 and 1208S To test the hypothesis that a more satisfactory balance between clinical acceptability and immunogenicity might be achieved with S. flexneri 2a purine auxotrophs containing fewer attenuating mutations, investigators at the Center for Vaccine Development compared the clinical acceptability and immunogenicity of CVD 1204 and 1208, which both carry deletions in guaBA, but CVD 1208 is also deleted in sen and set. Sequential groups of subjects were randomized to receive a single oral dose of CVD 1204 or CVD 1208 vaccine at 107, 108, or 109 CFU or placebo. Diarrhea, fever, or dysentery occurred in 30%, 22%, and 17% of CVD 1204 recipients, respectively (all doses combined), but in only 1 recipient (4%) of CVD 1208 (overall reactogenicity 35% vs. 4%, p ¼ 0.02). The respective anti-LPS immune responses to CVD 1204 and CVD 1208 were as follows: geometric mean IgA ASC were 445 and 62/106 PBMC, and proportion of subjects with fourfold rises in serum IgA and/or IgG was 100% and 71%, and proportion with fourfold rises in fecal IgA was 100% and 86%. In contrast to CVD 1204, fecal shedding of CVD 1208 ceased spontaneously in most subjects by day 7. The results show that whereas CVD 1204 was clearly attenuated compared with its wild-type parent (by retrospective comparison), it was still too reactogenic to serve as a live, oral vaccine. However, eliminating the ability to produce ShET1 and ShET2 significantly attenuates Shigella and provides convincing evidence that the ShETs are virulence properties in Shigella. Not only did CVD 1208 not cause diarrhea, dysentery, or high fever at high doses, but the 109 CFU dose elicited immune responses that one would predict are likely to be protective. To address regulatory concerns that prions could potentially contaminate vaccine products cultivated on animalcontaining media, CVD 1208 was reconstructed on animalfree media and designated as CVD 1208S. A phase I study in which subjects were randomized to receive a single oral dose of either placebo or CVD 1208S at 8- or 9-logs gave similar results to those reported for CVD 1208 (33). Moreover, IFN-g

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production by PBMC in response to Shigella antigens was observed in 57% recipients of 109 CFU (33). These data suggest that CVD 1208S retains a favorable safety and immunogenicity profile after reconstruction on animal-free media and shows promise as a live, oral Shigella vaccine. Phase II and phase IIb challenge trials with virulent S. flexneri 2a are planned for CVD 1208S.

Shigella Fundamentally Attenuated with a Mutation in virG S. flexneri 2a Harboring Deletions in virG/icsA and iuc (SC602) In 1990, Sansonetti and Arondel constructed a double mutant with deletions in both the virG/icsA gene and the iuc chromosomal locus (encoding the aerobactin iron binding siderophore) from S. flexneri 2a strain 454 (100). A transposon-generated deletion was created in the chromosomal gene iuc. The virG gene was inactivated using sacB suicide vector technology. The selected clone, designated SC602, harbored a deletion of the entire virG/icsA gene along with substantial flanking sequences totaling 10 kb. In initial phase I dose-response studies of SC602 at the U.S. Army Medical Research Institute for Infectious Diseases, 60% of subjects receiving 106 CFU experienced diarrhea, fever, and/or constitutional symptoms (Table 1) (27). In subsequent trials using 104 CFU, 20% of subjects experienced diarrhea or fever of brief duration (27,101). The peak geometric mean antiLPS IgA ASC response following 104 CFU was 25 per 106 PBMC; threefold or higher increases in serum anti-LPS IgA and IgG antibody were seen in 43% and 19% of subjects, respectively (27,101). The vaccine was detected in stool from all vaccinees, sometimes intermittently, for a mean of 12 days, though some shed for longer than four weeks. An efficacy challenge study conducted eight weeks after vaccination demonstrated that vaccination completely protected against fever and/or severe shigellosis (27). However, three of the seven challenged vaccinees (43%) experienced mild diarrhea (overall protective efficacy 50%) (Table 1). Vaccine recipients who were protected had more vigorous anti-LPS responses to vaccination (all had at least 75 IgA ASC per 106 PBMC and threefold rise in serum IgA antibody, and three of the four had threefold rise in serum IgG antibody) compared with unprotected vaccinees. In 2000, clinical trials of SC602 began in Bangladesh (A. Baqui, unpublished), an endemic area for S. flexneri 2a (102). Trials were conducted in adult volunteers, followed by children of 8 to 10 years, who received 104, 105, or 106 CFU of SC602 or placebo. Neither adults nor children experienced significant side effects, and SC602 was only rarely isolated from vaccinees. The final evaluation of SC602 in Bangladesh was a series of inpatient studies conducted in 12- to 36-month-old children who received escalating doses of 103 to 106 CFU. The vaccine was well tolerated. However, neither vaccine excretion nor immune responses were detected. The disappointing results of this attempt to use a minimally attenuated, live Shigella strain as a vaccine for children in the developing world suggests that safety and immunogenicity data from vaccine trials in adults of the developed world are not easily extrapolated to endemic areas. Perhaps lactoferrin in the intestines of milk-fed toddlers acted synergistically with the aerobactin (iuc) mutation to incapacitate the vaccine by depleting its iron stores. Maternal antibody, actively acquired immunity, and small bowel bacterial overgrowth may also mitigate intestinal colonization with a

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small number of Shigella even if the attenuation of the pathogen is minimal. S. sonnei Attenuated on the Basis of a Deletion Mutation in virG/icsA (WRSS1) The iuc mutation in SC602 provides little additional attenuation of S. flexneri 2a above that seen by inactivating virG/icsA alone (100); therefore, Hartman and Venkatesan developed a S. sonnei vaccine candidate (WRSS1) attenuated solely on the basis of a 212-bp deletion in virG/icsA created using sacB suicide vector technology described above for SC602 (67). A phase I trial was conducted at the Center for Vaccine Development in which subjects were randomized (double-blind) to receive either placebo or vaccine with buffer at a dose of 103, 104, 105, or 106 CFU (Table 1) (32). Self-limited fever and/or mild diarrhea occurred in 14%, 0%, 30%, and 33% of subjects in each ascending dose group, respectively. Similar dose-related trends in clinical tolerance were observed in a subsequent phase II trial conducted in Tel Aviv, Israel. One of the thirty subjects (3%) who ingested 103 or 104 CFU had diarrhea on day 1, which was followed by nausea and vomiting on day 5 (Table 1) (103). On the other hand, 27% of subjects who ingested the 5-log dose experienced diarrhea, and 13% had low-grade fever. Gastrointestinal symptoms graded as moderate or severe were reported by in 17% of those who received 103 or 104 CFU and in 33% of those who ingested 105 CFU. WRSS1 was excreted for an average of five days. More importantly, there was no microbiological evidence that WRSS1 spread to household contacts during a total of 192 days of exposure to colonized vaccinees (103). Vaccination elicited vigorous immune responses even at lower doses (Table 1). Following 104 CFU, the geometric mean titer of anti-LPS IgA ASCs was 73/ 106 PBMC, with a 95% response rate (10 ASC). In addition, 60% of these volunteers had  3-fold anti-LPS IgA serum antibody responses and 27% had a similar IgG response. Phase I and phase IIb S. sonnei challenge trials to assess the efficacy of WRSS1 are planned for an overseas site. S. dysenteriae 1 Attenuated by Deletions in virG/iscA and stxAB (WRSd1) Venkatesan et al. constructed WRSd1 from wild-type S. dysenteriae 1 strain 1617 by deleting the entire virG/icsA gene along with flanking sequences totaling 10 kb using suicide plasmid technology (104). The entire stxAB Stx gene was deleted by anaerobic growth on chlorate-containing medium, a technique that selects spontaneous deletion of the linked fnr fumarate and nitrate reductase regulator genes (60,105). Since fnr mutants of E. coli appear defective in colonization in vivo, this deletion probably further attenuates WRSd1. The candidate vaccine was administered to inpatient subjects at Johns Hopkins Center for Immunization Research in incremental doses ranging from 103 to 107 CFU (106). Overall, 8 of the 40 subjects (20%) developed diarrhea, 2 subjects (5%) passed a single dysenteric stool, and 2 subjects (5%) vomited. Anti-LPS IgA ASC responses were observed in 60% of subjects; in 20% the counts exceeded 40/106 PBMC. Fourfold or greater rises in anti-LPS IgA responses were observed in serum and stool in 25% and 44% of subjects, respectively. Fecal shedding of the vaccine strain was brief and detectable in only 22% of subjects. Interestingly, days of shedding but not dose of vaccine correlated with stronger antiLPS responses in several assays. Further clinical trials with WRSd1 have been abandoned in lieu of development of ‘‘second-generation’’ virG/icsA vaccines described below.

Second-Generation DvirG/icsA Shigella Vaccines Attenuated by Deletions in Enterotoxin Genes On the basis of the clinical trials with guaBA candidates described above, Venkatesan et al. sought to improve the safety profile of SC602 by incorporating set (ShET1) and sen (ShET2) enterotoxin deletions (107) using lambda Red recombineering (108). In addition to virG/IscA and enterotoxin deletions, strain WRSF2G11 was created with an msbB deletion incorporated into the Shigella virulence plasmid to further reduce intestinal inflammation caused by LPS (43). Second-generation vaccines incorporating these features also have been constructed in S. sonnei (WRSs3) and S. dysenteriae 1 (WRSd5) (109). Phase I trials of a second-generation S. sonnei vaccine are planned as an initial clinical evaluation of these new vaccines. S. dysenteriae 1 Attenuated in VirG/IcsA, Enterochelin, and Shiga Toxin Production (SC599) Sansonetti et al. constructed a S. dysenteriae 1 vaccine candidate with mutations in virG/icsA, stxA, entF (encoding the enterochelin iron-binding siderophore), fepA (encoding the surface receptor for enterochelin), and fes (release of iron from enterochelin). As a first step, a DstxA:HgR mutant was created from strain 7/87 by replacement of the stxA gene with a Hg resistance cassette to form SC595 (31). In an attempt to develop a virulent challenge strain model, SC595 was administered to subjects in doses ranging from 102 to 105 CFU. Generally mild illness was seen in 23% of subjects, suggesting the possible presence of other attenuating mutations (31). SC595 was further attenuated to construct the vaccine strain SC599 by deletion of virG/icsA, entF, fepA, and fes using the sacB strategy. A phase I trial of SC599 was conducted at the St. George’s Vaccine Institute in London in which 28 subjects received doses ranging from 102 to 108 CFU in bicarbonate buffer (70). Occasional gastrointestinal reactions and fevers were observed. Doses of 105 CFU or more consistently induced anti-LPS IgA ASC responses and occasional LPS-specific serum antibody responses. Next, a phase II trial was conducted at the CIC de Vaccinologie Chin-Pasteur and at the St. George’s Vaccine Institute (110). Subjects were randomized to ingest a single dose of placebo or SC599 at 105 CFU or 107 CFU. The respective frequency of reactions among the three groups was as follows: diarrhea (8%, 32%, and 14%), vomiting (5.4%, 5.3%, and 2.8%), and fever (2.7%, 10.8%, and 2.8%). In total, fecal shedding was found in 29% of vaccinees for approximately three to four days, beginning a mean of 8.6 and 3.7 days following 105 and 107 CFU, respectively. Some subjects did not manifest symptoms or fecal shedding for two to three weeks. These findings highlight the prolonged incubation period for S. dysenteriae 1 compared with other Shigella serotypes. The geometric mean anti-LPS IgA ASC responses to 105 and 107 CFU were 7.9 and 11.3/106 PBMCs, respectively, but exceeded 100 in several subjects. An IgA or IgG anti-LPS serologic response occurred in approximately 33% of subjects. Plans to proceed to vaccine trials in endemic areas are in progress.

SUBCELLULAR EXTRACTS OF VIRULENT SHIGELLA IpaB and IpaC invasins are readily extracted from cultures of virulent Shigella using hypotonic water treatment, and these proteins can be co-purified as a complex with LPS using ionexchange chromatography (111,112). This native proteinpolysaccharide complex (Invaplex) binds to mammalian cells

Chapter 64: Live and Subcellular Vaccine Strategies Against Shigella

and actively induces endocytic uptake of the Ipa proteins and LPS (unpublished). Oaks and Turbyfill have developed Invaplex as a candidate vaccine, demonstrating immunogenicity and efficacy for monovalent and bivalent products in the Sereny test (113). Preliminary data from clinical trials of S. flexneri 2a Invaplex (produced under current Good Manufacturing Practices) show excellent vaccine safety by the intranasal route and encouraging immune responses against both LPS and the native Invaplex antigen. A challenge trial with virulent S. flexneri 2a of volunteers immunized with Invaplex is in progress.

THE CONCEPT OF POLYVALENT SHIGELLA VACCINES Most Shigella vaccines developed to date are intended to elicit serotype-specific immunity as has been shown following virulent infection (6–10). However, it is not realistic to attempt to include all 50 Shigella serotypes in a vaccine; consequently, mechanisms for achieving heterotypic immunity have been sought. A strategy developed at the Center for Vaccine Development purports that 5 Shigella strains (S. sonnei, S. dysenteriae 1, and S. flexneri 2a, 3a, and 6) are most critical to include in a potential vaccine (71). Arguably, among the 14 serotypes belonging to S. dysenteriae, only type 1 warrants inclusion as a means of protection against the episodic occurrence of clinically severe pandemic disease (3–5,114). A S. dysenteriae 1 vaccine might also be useful for protecting populations at risk of exposure to agents of bioterrorism. S. sonnei (1 serotype) and S. flexneri (15 serotypes and subserotypes) are essential since they represent the most common causes of endemic shigellosis worldwide (1). As proposed by Noriega et al., it may be possible to include only three S. flexneri serotypes in a vaccine (2a, 3a, and 6) since these are likely to provide cross-protection against the remaining 11 S. flexneri serotypes and subtypes (11). Available studies suggest that combining antigens of multiple Shigella serotypes into a single parenteral or oral vaccine does not compromise immunogenicity of the separate components. The polyvalent vaccine prepared by Formal et al. containing S. flexneri 1b, 2a, and 3 plus S. sonnei protected monkeys against challenge with each homologous serotype (12). Finally, adults and children were successfully immunized with bivalent oral vaccines containing SmD S. flexneri serotypes 2a and 3 (18), S. flexneri 1 and 2a, or S. flexneri 3 and S. sonnei (10). These studies demonstrate in humans that protection against multiple Shigella serotypes with an oral polyvalent Shigella vaccine is feasible.

SHIGELLA AS A LIVE VECTOR Shigella Expressing Foreign Antigens Attenuated Shigellae are also attractive candidates to serve as live vector vaccines. These strains are delivered orally, colonize the FAE, and elicit a broad immune response that includes serum and mucosal antibodies, cell-mediated immune responses and a form of antibody-dependent cellular cytotoxicity (93,115); moreover, Shigella, which shares a high degree of homology with E. coli, is readily manipulated genetically. In theory, oral vaccines against a variety of infectious diseases can be developed by stable expression of foreign genes encoding protective antigens in a Shigella live vector strain. Initial uses of Shigella as a live vector employed DaroD S. flexneri strain SFL 124 as a carrier for antigens from S. dysenteriae including Stx B subunit and O-antigen determinants (116–118).

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Such strains elicited immune responses against both S. flexneri and S. dysenteriae antigens in animal models. Additionally, attenuated strains of Shigella have been used to express antigens from enterotoxigenic E. coli (ETEC) (119), VP4 of rotavirus (120), and the C3 epitope of VP1 of poliovirus (121). Immunization with these strains elicited immune responses in animal models against both the heterologous antigen or epitope as well as the Shigella vector. Engineering of specialized stabilized plasmids have enabled reliable, high-level expression of multiple antigens from a single plasmid in attenuated Shigella strains. At the Center for Vaccine Development, investigators have used this system to express multiple different ETEC fimbriae as well as LT antigens in DguaBA S. flexneri vaccine strains that can elicit both serum and mucosal immune responses to the heterologous antigens as well as to the Shigella vector in animal models (122–126). A phase I trial of a CVD 1208S(pCFA/I-LThA2B) is in progress at the Center for Vaccine Development. These systems demonstrate the possibility of constructing a multicomponent vaccine formulation capable of generating broad spectrum immunity against two important pathogens.

DNA Delivery The ability of Shigella to invade eukaryotic cells and gain access to the cytoplasm has been exploited for the delivery of DNA vaccines. Sizemore and coworkers (127) used the marker protein b-galactosidase, driven by the CMV promoter, to show that mucosal administration of an attenuated strain of Shigella could deliver a DNA plasmid to the cytosol of a eukaryotic cell and elicit immune responses in a mouse model. Subsequently, investigators have used attenuated Shigella strains to deliver DNA plasmids encoding fragment C of tetanus toxin (128), measles virus H and F antigens (129,130), and HIV gp120 (131). In each case immune responses to the encoded antigen were generated in immunized animals.

CONCLUSIONS The increasing knowledge of the Shigella’s genome has allowed investigators to target diverse genes for deletion mutations in an attempt to construct safe, attenuated oral Shigella vaccines. Experience suggests that the major impediment to creating a successful vaccine will be achieving safety in industrialized countries while retaining immunogenicity in developing countries. In the near future, it is expected that several promising vaccine candidates will reach phase I, phase II or phase IIb clinical trials. The mutation or combination of mutations that demonstrates the best safety without compromising immunogenicity will likely be reproduced in other serotypes to form a polyvalent Shigella vaccine containing the most prevalent serotypes worldwide. It is possible that such a Shigella vaccine can also serve as a live vector vaccine to express antigens that confer protection against other relevant enteric pathogens.

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65 Vaccines Against Enterotoxigenic Escherichia coli Ann-Mari Svennerholm Gothenburg University Vaccine Research Institute and Department of Microbiology and Immunology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden

Gregory M. Glenn Intercell USA, Inc., Gaithersburg, Maryland, U.S.A.

INTRODUCTION Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in the developing world (1–4). On an annual basis, it has been estimated that ETEC causes 280 to 400 million diarrheal episodes in children below five years and an additional 400 million cases per year in persons above five years in low- and middle-income countries (LMIC) (2). In addition, ETEC has been shown to be an important cause of diarrhea during natural disasters such as flooding and earthquakes (1,5). ETEC is the most frequent cause of all diarrhea in travelers, resulting in 20% to 75% of all such cases (1,6). The illness caused by ETEC usually lasts from three to five days and ranges from mild diarrhea without dehydration to severe cholera-like disease (1). Children in developing countries may experience one to three episodes of ETEC diarrhea per child per year during their first years of life and repeated episodes of diarrhea may contribute to malnutrition in these children (1,7). In regions of the world where ETEC is highly endemic, there is a decline in ETEC diarrhea incidence with age, with peaks observed in children 6 to 18 months of age (1,3), whereas there is no evident association with age in short-time visitors to endemic areas (1,2,8). However, the incidence of ETEC rapidly decreases in persons from developed countries during prolonged visits to ETEC-endemic areas (1). These observations strongly support the hypothesis that effective immunity may develop after ETEC infections, and consequently protection by way of an ETEC vaccine is achievable. In this chapter we describe different efforts that have recently been attempted or that are in progress to develop effective ETEC vaccines both for use in children in LMIC and in travelers to ETEC-endemic areas. These efforts are to a large extent based on knowledge of the key pathogenic and immune mechanisms in ETEC disease.

PATHOGENIC MECHANISMS ETEC colonizes the mucosal surface of the small intestine and causes disease by producing one or more enterotoxins that exert their action on the epithelial cells. These toxins include a heat-labile toxin (LT) which is structurally, functionally and antigenically very similar to cholera toxin (CT), consisting of an active A subunit and five identical binding subunits (LTB) which both share about 80% homology with corresponding

CT subunits (CTB and CTA) (9). Immunity against LT is to a large extent directed against the B subunits (9). ETEC may also produce heat-stable enterotoxin (ST), which exists in two variants, STp and STh, which are small molecular weight peptides consisting of 18 and 19 amino acids, respectively. ST is not antigenic unless coupled to a carrier protein and immune responses against ST are not induced after infection with ST producing ETEC (1,9,10). The relative proportion of strains producing LT and or ST varies from one geographic area to another, as well as in patients with diarrhea and in asymptomatic carriers (1,2,4). Some epidemiological studies have shown that ca 50% of clinical ETEC isolates produce ST only, whereas LT-only strains have predominated in other areas (1,11). However, despite the conduct of many high-quality studies to define the epidemiology of ETEC and toxin distribution, it is generally recognized that methods for toxin detection have been difficult to establish in many laboratories and study methods vary, bringing uncertainty to the description of toxin distribution in ETEC strains. As a result, several efforts are underway to improve the descriptive epidemiology of ETEC disease. For example, a large study on the cause of hospital-based diarrheas in children in different countries in Africa and Asia is presently in progress using predominantly molecular diagnostic methods of detection, and may reveal the relative importance of ETEC, including the role of strains with different toxin and CF profiles, as a cause of severe diarrhea in children in these regions. Additional studies are in progress, for example, in Latin America. ETEC bacteria colonize the small bowel by means of one or more colonization factors (CFs), which usually are fimbriae or fibrillae (1,12). More than 22 different CFs on human ETEC have been described (12,13) and additional ones have been recognized (Savarino et al., unpublished) or will most likely be identified. Among the wide range of CFs, CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, and in some studies also CS7, CS14, CS17, CS19 and CS21 are the ones most frequently expressed by clinical ETEC isolates (1,10). Several of the CFs may be expressed on the same bacteria, for example, CS1 þ CS3, CS2 þ CS3, CS4 þ CS6, CS5 þ CS6, although several strains may express CS6 alone. Some CFs, for example, CS6 and CS21 (Longus), may only be expressed genotypically and not phenotypically on the bacterial surface of certain ETEC strains (14,15), which may preclude their roles as CFs in

Chapter 65: Vaccines Against Enterotoxigenic E. coli

vivo and their importance as targets for a CF-based vaccine. Together, the most prevalent CFs have been found on roughly 50% to 80% of all ETEC strains (1,10,12) although in variable frequencies in different geographic areas, during different seasons and in different categories of patients. Furthermore, the most common CFs have been shown to be considerably more prevalent on diarrheagenic strains than on ETEC isolated from nondiarrhea stool specimens (3). Most of the CFs are composed of up to 100 identical structural subunits and several of the CFs also express distinct tip proteins. Both the structural proteins and tip proteins have been considered as candidate immunogens (10,12,13). Recent studies have also revealed that antibody preparations against whole CFA/I fimbriae as well as the CFA/I tip protein in milk formulas have afforded significant protection in human volunteers challenged with virulent CFA/I ETEC (16). Immunization using recombinantly produced CF tip proteins has also afforded significant protection against challenge with ETEC expressing corresponding CFs in primates (Savarino et al., unpublished). Some of the CFs are immunologically related, as in the CF I-like group (including CFA/I, CS1, CS2, CS4, CS14, CS17, CS22 and PCFO71) and the coli surface 5–like group (with CS5, CS7, CS18 and CS20) (13). Within these groups, cross-reactive epitopes have been identified both among the structural subunits (17) and on the receptor binding tip proteins of the CFs. The tip proteins of the CFA/I group have been shown to be highly conserved and capable of inducing strong immune responses not only against the homologous CF but also against ETEC expressing other CFs within the same group (13). Previous studies have also shown that the N-terminal of the structural subunits of the CFA/I group are highly conserved. Thus, monoclonal antibodies against the N-terminal CFA/I structural subunit have prevented binding of ETEC expressing CS2, CS4 and CS14 (all CFA/I group fimbriae) to intestinal epithelia cells (Caco-2 cells) and afforded significant protection against challenge with ETEC expressing CFA/I as well as CS4 in experimental animals (17). Such epitopes or structures, both on the tip and structural subunits of CFs that may react with immunologically cross-reactive antibodies, have been recognized as promising candidate antigens for inclusion in an ETEC vaccine providing broad protective coverage (10,13,17). More than 100 different O groups of E. coli have been identified among clinical ETEC isolates (11,18). In addition, rough strains that are nontypeable with regard to O-antigen are not uncommon (1,11). Although there are certain ETEC serogroups that are more prevalent than others, there are large geographic differences. Still, a vaccine containing common O-antigens may result in increased protective coverage, since some studies in experimental animals suggest that antibodies against certain E. coli O-antigens may protect against ETEC of homologous serogroups (10).

IMMUNE RESPONSES AGAINST NATURAL ETEC’DISEASE Studies in Challenged Volunteers Specific antibodies locally in the intestine, both antibodies locally produced and most likely also antibodies derived from the circulation, have been considered as the most important mediators of immune protection against ETEC (1,9,10). Hence, there are reports showing a direct relation between systemic as well as mucosal antibody levels against CFs and

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protection against virulent ETEC expressing corresponding CFs in human volunteers challenged with fully virulent ETEC strains. For example, in a recent study in adult American volunteers, although the vaccine was not protective, higher serum anti-CS3 IgA titers correlated with less severe diarrheal illness (19). Similarly, IgA serum antibody levels against LT that seem to be associated with protection have been identified in human volunteers challenged with LT producing ETEC (Bourgeois et al., to be published).

Immune Responses to Natural ETEC Disease The low rates of ETEC disease observed in children above two years in high endemic areas (1,2,4) suggest that protective immunity will develop in response to natural infections. To analyze the nature of such responses, over 300 children in an urban slum area in Dhaka, Bangladesh were followed from birth up two years of age for incidence of initial and reinfection with ETEC of the same or heterologous ETEC phenotypes. Interestingly, none of the children who had experienced diarrhea due to CFA/I, CS1 þ CS3, CS2 þ CS3 or CS5 þ CS6 expressing ETEC had a repeat episode of diarrhea from an ETEC strain expressing homologous CFs during the two-year study period but they experienced frequent diarrheal episodes caused by ETEC strains expressing heterologous fimbriae, suggesting the development of immunity against the CFs (3). Similarly, asymptomatic infections with ETEC expressing these CFs were not followed by symptomatic infections with ETEC expressing homologous CFs, whereas repeated infections with CS6 only and CF-negative LT-only ETEC were not uncommon (3). In accordance with these findings, no association was found between anti-LT titers and the risk of developing LT ETEC disease in a study in children in a high endemic area in Egypt (20). The relatively poor protection afforded by LT produced by ETEC during natural infection or disease may be explained by the fact that LT is often produced in comparatively low amounts during infection in the gut, and immune responses are often rather modest as compared with after administration of CTB or LT in different candidate vaccines (9,10). For example, in an inpatient ETEC challenge study, anti-LT immune responses in naı¨ve subjects in whom >90% became ill were approximately 10-fold lower than vaccinees receiving an LT patch (21) (Fig. 1). Findings noted above from Bangladesh and Egypt are at variance with observations from a birth cohort in Guinea Bissau (28) in which LT, but not the CFs, were reported to contribute to protection against reinfection. However, this conclusion was based on estimates of protection against asymptomatic infections. Asymptomatic infections, particularly LT ETEC infections, may play an important role in the pathogenesis of diarrhea by disrupting normal mucosal barriers (Glenn et al., to be published), the corollary being that toxin immunity would prevent such disruption, which lowers the threshold for symptomatic disease. Further support for a protective role of CF antibodies against natural ETEC disease comes from results of a casecontrol study in 397 Egyptian infants showing that serum IgG antibody titers against CFA/I were inversely related to the risk of developing CFA/I ETEC diarrhea in children <18 months (20). However, it cannot be excluded that these serum antibody levels merely reflected mucosal immune responses or spillover of locally produced antibodies into the circulation, since mucosal immune responses were not measured in these studies. Patients hospitalized with ETEC diarrhea develop significant

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Figure 1 Anti-LT response to the heat-labile toxin of ETEC (LT): comparative superiority of anti-LT responses due to patch application versus live organism oral challenge. Patches containing LT or placebo were applied to the upper arm on days 0, 21 and 42, and subjects were challenged orally with ETEC on day 56. Serum antibody levels were measured on days 0, 21, 42, 55, and 3 weeks post challenge (day 77), and expressed above as geometric means of ELISA units with 95% CI. Abbreviations: ETEC, enterotoxigenic Escherichia coli ; LT, heat-labile toxin.

IgA antibody responses in the intestine or intestine-derived antibody-secreting cell (ASC) responses against the CFs of the infecting strain. These responses may be associated with clearance of the infection (1,23,24). Mucosal immune responses against cross-reactive CFs have also been induced in patients infected with CF-positive ETEC (25). These findings give hope that a CF-based ETEC vaccine composed of the six to seven most common CFs may provide protection against ETEC expressing the homologous but also immunologically related CFs. Whereas the antitoxic immunity that develops in response to natural infection may be relatively ineffective, antitoxic immune responses induced by some forms of vaccination may play an important role in protecting against LT producing ETEC. Multiple field trials using the CTB as antigen as well as the native LT antigen have shown that anti-LT toxin immunity alone can elicit protection in both children from endemic areas and travelers (6,22,26–29). LT antigen has also been shown to cooperate synergistically with CFs in inducing protection against ETEC strains producing LT and expressing homologous CFs (10).

ETEC CANDIDATE VACCINES A CF-based ETEC vaccine should contain CF antigens present on the most prevalent ETEC pathogens, in particular on strains producing LT þ ST or ST alone, to provide broad-spectrum protection. Thus, a multivalent, oral ETEC vaccine containing CFA/I, CS1-6 and/or corresponding CF tip proteins and an LT toxoid may have the potential to provide protection against ca 80% of ETEC strains worldwide (10,30,31). If an effective ST toxoid could be developed and added, the protective coverage could certainly increase even further. The vaccine should provide strong mucosal immunity against the key protective antigens locally in the small intestine, since the local antibody response in the gut seems to best reflect the protective immune response (30,31). Alternatively, or in addition, the vaccine may induce high levels of systemic immunity that may contribute to protection, through active transport into the gut (21) or transudation of serum antibodies into the small intestine. Different strategies in the form of inactivated or live candidate vaccines have been taken to deliver ETEC CFs and toxin antigens to the human immune system to elicit protective immune responses (Table 1).

Table 1 Inactivated candidate ETEC Vaccines in Development ETEC vaccine antigens

Route

Toxoids (CTB, LT, mutant LT, LTB, CTB/LTB hybrid) Inactivated CF-expressing whole bacterial cells þ toxoid Purified CFs or CF tip proteins Heat-stable enterotoxin toxoids

Oral or transcutaneous Oral Oral or transcutaneous Oral or transcutaneous

Abbreviations: ETEC, enterotoxigenic Escherichia coli; CF, colonization factor; LT, heat-labile toxin; CTB, cholera toxin subunit B; LTB, heat-labile toxin subunit B.

Inactivated ETEC Vaccines Enterotoxoids There are several candidate antigens/toxoids that are already in use or under development to provide immunity against LT. They include CTB, LTB, CTB/LTB hybrid toxoids or fully active LT (Table 1). Since the nontoxic LTB as well as CTB subunit components are strongly immunogenic, stable in the gastrointestinal milieu and capable of binding to the intestinal epithelium, they are both suitable candidate antigens to provide anti-LT immunity (9). Oral CTB has also afforded significant, 50% to 70%, protection against E. coli LT disease caused by LT-only as well as LT/ST-producing ETEC, both in persons living in ETEC-endemic areas and in travelers to such countries (8,10,30). This protection has been short-lasting (three to six months) or only tested for protection during limited periods, as in travelers. To date, the oral inactivated cholera vaccine, Dukoral1, containing 1 mg recombinantly produced CTB per dose is licensed in more than 20 different countries for use as a prophylactic vaccine against LT (and ST/LT) producing ETEC. The vaccine should be given in two oral doses with bicarbonate buffer one-two weeks apart, with the second dose given at least one week before possible exposure to LT ETEC. It has been suggested that an LT toxoid may be slightly more effective than CTB in inducing protective anti-LT immunity based on the presence of unique epitopes on the LTB molecule (9). Alternatively, hybrid CTB/LTB molecules that contain both shared and unique LTB as well as CTB epitopes (32) may be used to provide protection both against cholera and ETEC, since both

Chapter 65: Vaccines Against Enterotoxigenic E. coli

these infections are prevalent in young children <2 years, for example, in West Bengal (33). Such a hybrid CTB/LTB toxoid will be evaluated for safety and immunogenicity against CTB as well as LTB in clinical trials intiated 2009. Transcutaneous Immunization with LT Antigen The safety and immunogenicity as well as protective capacity of transcutaneously administered E. coli LT have been extensively tested in human volunteers given two or three immunizations with 37.5 to 50 mg native LT in patches applied on the skin surface at two- to four-week intervals (34–38). Studies in healthy adult American volunteers have shown that such LT patches do not give rise to any significant systemic side effects such as diarrhea and induce only modest local reactions such as pruritus, rash and infrequent transient pigment changes on the skin (34,35). LT patches have been shown to be strongly immunogenic, resulting in significant serum IgA and IgG as well as IgA ASC responses against LT in almost 100% of the immunized volunteers (Fig. 1) (36). In an initial study in healthy North American volunteers immunized at day 0, 21 and 42 with 50 mg of LT delivered transcutaneously, the attack rate for diarrhea was not significantly diminished in the vaccinated volunteers versus controls. Nonetheless, the vaccinees had a significantly longer time to onset of diarrhea, significantly fewer and smaller stools and needed intravenous rehydration less frequently. This initial study was followed by a double-blind, placebo-controlled field trial in adult travelers to Mexico or Guatemala, in which promising results were obtained. Administration of two patches with 37.5 mg LT in each given two to three weeks apart afforded highly significant protection (PE 75%, moderate/severe diarrhea, p < 0.01) against travelers’ diarrhea of any origin (37). Interestingly, this underpowered study did not show significant protection against ETEC disease (PE 66%, p ¼ 0.13). However, in analogy with findings in challenged volunteers, the vaccinated travelers had significantly shorter duration of ETEC diarrhea (2.2 vs. 0.45 days, p ¼ 0.020) and lower mean stool frequency (10.5 vs. 4.3/ day, p ¼ 0.038) than the placebo controls. The explanation for the observed protection against non-ETEC travelers’ diarrhea needs to be further explored in future studies, but is consistent with both field studies demonstrating toxin immunity generated by CTB (6,22,26) and with blocking the nonspecific enhancing effects by LT on colonization of non-LT ETEC (39). Collectively, these results indicate a protective effect of transcutaneous immunization with LT, and that the response to LT in a patch extends protection beyond ETEC infections. Additionally, the dry patch formulation represents an ambient temperature-stable, needle-free format potentially suitable for use for children in the developing world (40). In light of the field efficacy data in travelers, the suitability of a patch and simplicity of the vaccine construct, there are proponents urging that this ETEC vaccine approach should be evaluated in infants and children in the developing world. Heat-Stable Enterotoxin Toxoids Attempts to prepare a suitable ST toxoid have so far not been successful, partly because of the small size and high cysteine content of the ST molecule (6 of the 19 amino acids in STh are cysteines, which results in extensive folding of the ST peptide) (9,30). Coupling of ST or shorter ST peptides, for example, a decapeptide, to different carrier proteins, for example, CTB or CFs, by chemical or recombinant techniques (9,10) has resulted in ST immunogenic conjugates, but with retained toxicity.

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Furthermore, because of the small molecular size of the ST molecule, comparatively high levels of anti-ST antibodies will be required to neutralize the toxin on a molar ratio. To our knowledge, no nontoxic yet immunogenic ST conjugate is presently available that can be reproducibly produced but efforts are in progress to construct such conjugates in different laboratories (10,30). One alternative possibility that may be explored is to administer immunogenic ST conjugates by the transcutaneous route; some residual toxicity may be acceptable for such constructs since toxin-active LT can be delivered safely by this route (35–37). Purified Colonization Factors Purified CFs have drawbacks as oral immunogens, since they are sensitive to proteolytic degradation and expensive to prepare (10,30). To protect the CFs from degradation in the stomach, immunization with purified CFs incorporated into biodegradeable microspheres has been attempted (43) but significant protection was not induced against subsequent challenge with ETEC expressing the homologous CFs, either when immunizing with high doses of a combination of CS1 and CS3 or recombinantly produced CS6 (30,43). Alternative approaches include administering the CFs transcutaneously. For example, E. coli CS6 has been given incorporated into patches on the surface of the skin, alone or together with nonmutated LT (42). When administering comparatively high doses of CS6 in such patches, no response to CS6 was observed in the absence of LT (which is also a strong adjuvant). However, combined administration of CS6 and LT induced antibodysecreting cells (ASC) as well as serum antibody responses against CS6 in about half of the volunteers and serum anti-LT responses in all of them (43). An additional approach to provide anti-LT and CF immunity by purified proteins includes the production of chimeras consisting of ETEC adhesin and enterotoxin B subunit where the tip of CFA/I is linked to LTB or CTB. These chimeras were found to be bifunctional and capable of eliciting both antitoxin and anti-adhesin immune responses (41). Inactivated Whole Cells Another approach to provide CFs in an ETEC vaccine is to prepare killed ETEC bacteria that express the most important CFs in immunogenic form on the bacterial surface (10,30). Such inactivated bacteria may be combined with an appropriate toxoid. Inactivation of the bacteria may be achieved by treatment with formalin or colicin E1 (30), which has resulted in killing of the bacteria without significant loss in antigenicity of the different CFs and O-antigens (10). Thus, CFs on ETEC bacteria inactivated by mild formalin treatment have been shown to be more stable than purified CFs in the gastrointestinal milieu as well as to retain their immunogenicity, fimbrial structure and capacity to bind to eukaryotic cells. The ETEC vaccine most extensively studied in clinical trials, rCTB-CF ETEC, consists of a combination of recombinantly produced CTB (rCTB) and formalin-inactivated ETEC bacteria expressing CFA/I and CS1-CS5 as well as some of the most prevalent ETEC O-antigens (10,30). This vaccine has been shown to be safe and to elicit significant IgA immune responses locally in the intestine in a majority (70–90%) of Swedish vaccinees (36). Phase I and II trials in Swedish, Bangladeshi and Egyptian adult volunteers have shown that the vaccine is well tolerated and gives rise to mucosal immune responses, that is, immune responses in intestine (gut lavage fluid) or

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peripheral blood ASCs against the different vaccine CFs in 70% to 100% of the vaccinees (23,44–46). Furthermore, the vaccine has been shown to induce comparable immune responses against the CFs and LT locally in the intestine as clinical ETEC disease (23). In safety and immunogenicity clinical trials in children in LMIC (47–49), the rCTB-CF vaccine was found to be equally immunogenic as in adults (23,44–46), and well tolerated, except in the youngest infants and toddlers. Since increased frequency of vomiting was observed in children 6 to 17 months of age in Bangladesh (49), a dose finding study was initiated. This study showed that a quarter of a full dose of rCTB-CF ETEC vaccine was equally safe and almost as immunogenic in the youngest infants as a full dose in older children (49). In an initial pilot study in Austrian travelers going to different countries in Asia, Africa and Latin America, the CTBCF ETEC vaccine given in two oral doses conferred 82% protective efficacy (PE; p < 0.05) against ETEC diarrhea (50), but the number of ETEC diarrhea cases fulfilling the inclusion criteria was low. This study was followed by two larger placebo-controlled phase III trials in American travelers going to Mexico and Guatemala to assess the protective efficacy of the rCTB-CF ETEC vaccine (51). The first study, encompassing nearly 700 volunteers, did not meet primary endpoints but the vaccine provided significant protection (PE 77%; p = 0.039) against nonmild ETEC diarrheal illness, defined as symptoms that interfered with the travelers’ daily activities or more than five loose stools in a day. However, no significant protection was observed against ETEC diarrhea of any severity, including mild cases (51). The subsequent equally sized trial in the same setting also revealed that the vaccine protected against more severe symptoms in those vaccinees in which vaccine take could be documented (52). The only pediatric study to assess efficacy of the rCTB-CF ETEC vaccine has been undertaken in 350 children 6 to 18 months of age in rural Egypt (30; Savarino et al., unpublished). In that double-blind, placebo-controlled trial in which disease detection was based on active surveillance through semi-weekly household visits and cultures of fecal specimens from children with diarrhea, no significant protection was conferred by the vaccine (PE = 20%). In part, it could be that because active surveillance for diarrhea was undertaken, most cases were relatively mild; this is known to result in lower protective efficacies, for example, as has been described for recently licensed rotavirus vaccines (53), as compared with when passive surveillance is performed and protection against moderate to severe dehydration is determined. It may also be explained by the finding that the young children participating in the trial in Egypt seemed to respond less well immunologically to the vaccine than similarly immunized older children and adults in the same setting (46,47) and in Swedish and American adults (10,45,51). This finding is in agreement with observations for several other oral vaccines, for example, poliovirus, rotavirus and Shigella experimental vaccines, all of which have been shown to be less immunogenic in infants and young children in the developing world, than in adults or children in industrialized countries. Further Development of rCTB-CF ETEC Vaccine On the basis of the results from testing the rCTB-CF ETEC vaccine in children, studies to improve its efficacy are in progress. These efforts include increasing the amounts of protective antigens in the vaccine, in particular, increasing the

expression of CFs on the bacterial surface by recombinant technology (10,54). To examine the feasibility of this approach to express high quantities of CFA/I, which is one of the most prevalent CFs, on the surface of bacteria, the entire CFA/I operon containing four genes cfaA, cfaB, cfaC, and cfaE, was cloned into plasmid expression vectors containing the powerful tac promoter, which is under the control of the lacIq repressor present on the plasmids. By using this approach, CFA/I was expressed in considerably higher quantities on the surface of E.’coli K12 bacteria than on previous vaccine strains, as determined by different immunoassays, that is, a quantitative dot blot assay and inhibition ELISA, and as shown by immunoelectron microscopy (54). Similarly, by using a related approach, CFA/I fimbriae could be expressed in high quantities on the surface of a nontoxigenic strain of Vibrio cholerae (54). Indeed, the recombinant E. coli strain expressed up to 10-fold higher levels of CFA/I fimbriae compared with the CFA/I-positive strain that was used in the original rCTB-CF ETEC vaccine; the latter strain had previously been shown to be among the highest natural producers of the CFA/I fimbriae among >100 tested wild-type ETEC strains. Oral immunization of mice with formalin-killed bacteria of the CFA/I overexpressing E. coli strain induced significantly higher serum IgA antibody responses compared with the old vaccine strain (54). On the basis of this promising approach, work has been initiated to overexpress other prevalent ETEC CFs on the surface of E. coli K12 or nontoxigenic ETEC strains. By using the recombinant approach, a nontoxigenic strain has also been constructed that can overexpress the nonfimbrial CS6 protein, which is expressed in comparatively low levels on natural clinical ETEC isolates (Svennerholm A, unpublished). The recombinant construct expresses >20-fold higher quantities of CS6 (55) than the corresponding strains used in the original rCTB-CF ETEC vaccine, which did not provide CS6 immunity (10). Alternative methods have also been established that allow inactivation of CS6 positive strains with retained CS6 antigenicity since this protein is sensitive to formalin treatment. Work is also in progress to construct a series of recombinant strains that express the additional most prevalent CFs, alone or in combination, on nontoxigenic E. coli or ETEC strains. Other efforts to improve the efficacy of the rCTB-CF ETEC vaccines include usage of an alternative LT toxoid, for example, a hybrid LTB/CTB toxoid (33). They also include evaluation of the capacity of different putative mucosal adjuvants, for example, LT-based adjuvants (56), in particular a double-mutated LT molecule, LTR192G/L211A, which has been shown to be safe and to have strong adjuvant activity in experimental animals (57). Studies are also planned to give the vaccine by different routes, for example, by the sublingual route, which has recently been shown to be a very simple and efficient administration route for mucosal vaccines (58).

Live Oral ETEC Vaccines The potential of live ETEC vaccines as a tool for protection against diarrhea was initially demonstrated by early findings by Levine et al. in human volunteers that a live vaccine strain expressing CS1 and CS3 fimbriae, but lacking the genes encoding LT and ST, induced 75% protection against challenge with wild-type ETEC expressing corresponding CS factors as well as LT and ST (8,30). Different strategies have thereafter been explored in which attenuated ETEC, Shigella, V. cholerae, or Salmonella expressing different CS components alone or in

Chapter 65: Vaccines Against Enterotoxigenic E. coli Table 2 Live Attenuated ETEC Candidate Vaccines Under Development Vaccine approach or prototypes

Developer

Attenuated nontoxigenic ETEC bacteria expressing colonization factors Hybrid Shigella/ETEC live vector vaccine

ACE Biosciences

Vibrio cholerae Peru 15 expressing cholera toxin subunit B Salmonella expressing heatlabile toxin subunit B Vibrio cholerae CVD 103-HgR expressing CS3

Center for Vaccine Development, University of Maryland, Maryland, U.S.A. Avant Immunotherapeutics

Emergent Biosolutions

719

evaluated in phase I studies for mucosal immune responses to different CFs with promising results. It was found that all three vaccine strains were safe and induced significant mucosal immune responses against CFA/I and CS1-3 without inducing proinflammatory responses. Additional attenuated ETEC strains, with deletion of the above three genes, have recently been constructed. These strains, ACAM2023, ACAM2025, ACAM2027, express LTB with CS4CS6, CFA/I and CS1CS3, respectively (8,30). Studies have been initiated to prepare a dry formulation of these strains as well as to test a cocktail of all 3 strains for safety and immunogenicity and protective efficacy against challenge in adult American volunteers (Bourgeois A, personal communication).

Berna Biotech/Crucell

Abbreviation: ETEC, enterotoxigenic Escherichia coli ; CS, coli surface antigen.

combination with an LT toxoid have been constructed as putative vaccine candidates (Table 2) (8,30). Genetically Attenuated ETEC Strains as Live Oral Vectors A promising approach that has been extensively evaluated utilizes attenuated ETEC strains as vectors of key ETEC protective antigens. Such strains have previously been developed (30,59), for example, PTL002 and PTL003, which are toxinnegative derivatives of an O6:H16 prototype CS1 þ CS3 vaccine and harbor a mutation in aroC (required for biosynthesis of aromatic amino acids). PTL002 has an additional mutation in ompR, while PTL003 has mutations in ompC and ompF (which code for outer membrane porins). When given in a single dose of 5  109 colony forming units (CFUs) to adult volunteers, both strains were found to be safe and immunogenic, but significantly more recipients of PTL003 exhibited fecal shedding and mounted more robust serum antibody and IgA anti-CF ASC responses (59). PTL003 was shown to induce immune responses against CS1 and CS3 of comparable magnitude as the corresponding wild-type strain (60). When PTL003 (given in two doses, each containing 2  109CFU) was evaluated for protection against challenge with a wild-type CS1 þ CS3 positive ST/LT ETEC strain, neither the attack rate for diarrhea nor total stool volume was significantly diminished in vaccinees versus placebo recipients (19,60). It was speculated that the lack of protection might be explained by use of too low a dose for immunization and too high a challenge dose. Hence, it has been recommended to identify lower challenge doses of virulent ETEC that can be used to assess protective efficacies in challenge studies (19,30). Further developments of nonpathogenic E. coli strains expressing CFs include construction of ACAM2010, which is a similar vaccine candidate strain derived from wild-type WS1858B (O71:H, LT /ST þ, CFA/I, astA) by deleting STh (encoded by estA), EAST1 (enteroaggregative E. coli enterotoxin 1 encoded by astA), aroC ompC and ompF (61). In a small phase I clinical trial, the CFA/I expressing strain ACAM2010 was well tolerated and most recipients of a 109 CFU dose excreted the vaccine strain and had serum IgG and IgA ASC responses to CFA/I. In another study, two additional different genetically modified ETEC candidate vaccines have been generated, which are attenuated ETEC strains with defined deletion mutation in aroC, ompC and ompF, and express CS2CS3 (ACAM2007) or CS1 þ CS2 þ CS3 (ACAM2017) (62). The strains have been

Multivalent Shigella/ETEC Vaccines An alternative approach that has been successfully explored is to construct Shigella strains that are attenuated compared with their wild-type parent strains and to use these strains as live vectors to express ETEC antigens (8,30,63). Enlarging on this strategy, different live Shigella based multivalent Shigella/ETEC hybrid vaccines have been constructed wherein the important fimbrial CFs are expressed along with mutated LT in attenuated Shigella (56). An ambitious project is underway in which five attenuated Shigella serotypes (S. dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6 and S. sonnei) have each been engineered to carry stable expression plasmids that encode various ETEC fimbriae and the LTh B subunit (8). The five Shigella live vector strains in the vaccine will collectively express CFA/I, CS1-6, and genetically detoxified heat-labile toxin from a human ETEC isolate. Combinations of several attenuated Shigella strains expressing various ETEC fimbriae and LTB or a mutant LT adjuvant, LThK63 (63), have been shown to be well tolerated and to stimulate systemic IgG and mucosal IgA immune responses to both the ETEC and Shigella antigens in preclinical models. Studies are in progress to evaluate one of the five live vector strains, that is, attenuated S. flexneri 2a strain CVD 1208S expressing CFA/I and LTh B subunit, in dose-escalating phase I clinical trials to assess the live vector’s clinical acceptability and mucosal immunogenicity in humans (8). Attenuated Vibrio cholerae and Salmonella Strains as Vectors for ETEC Antigens A number of other live oral vaccine prototypes have been developed and evaluated for safety and immunogenicity in phase I clinical trials in recent years. They include attenuated Salmonella serovar Typhi and V. cholerae O1 strains expressing CTB/LT B subunits and different ETEC CFs (8,30). For example, CTB has been overexpressed ca 30-fold compared with the original live attenuated V. cholerae strain, Peru-15 (64). Experimental animals immunized orally with this CTB overexpressing strain have responded with high levels of anti-CTB titers. This B subunit overexpressing strain has been suggested as a candidate bivalent cholera/ETEC vaccine, since it was capable of inducing antitoxin antibody levels that neutralized both CT and LT in tissue culture assays (64). Different ETEC CFs have also been cloned into V. cholerae and Salmonella vaccine strains. Using immune electron microscopy it was shown that CFA/I can be expressed as fimbriae by an attenuated V. cholerae strain (54), and that, for example, CFA/I and CS3 can be surface expressed by the Salmonella Ty21a vaccine strain and CS3 by the live oral CVDHgR cholera vaccine strain (65,66).

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Enhancement of Vaccine Immune Response in Children in Developing Countries On the basis of the findings for most oral vaccines of lower immune responses and protective efficacies of oral vaccines in children in developing countries than in older age groups in the same regions and in Westerners, studies may also be undertaken to evaluate different approaches to enhance the immunogenicity of oral vaccines in young children in LMIC (67). These approaches include breast-milk withdrawal at the time for oral vaccination as well as pretreatment of children with micronutrients, for example, zinc and other micronutrients and/or antiparasitic treatment before vaccination. Indeed, very promising results have recently been obtained showing that Bangladeshi children 6 to 18 months of age developed significantly stronger antibacterial immune responses to orally given cholera vaccine Dukoral, which was used as a model vaccine, when breast feeding was withheld for three hours before vaccination or when the children were provided zinc supplementation three weeks before vaccination (68). The possible benefits of additional modifications, such as administering the vaccines in alternative buffers or formulations or through avoidance of the intestinal milieu altogether by use of an LT patch, may be further evaluated in infants and young children in developed and developing countries (67).

EVALUATION OF CANDIDATE VACCINE STRAINS Testing of new candidate vaccines for protective efficacy in preclinical trials is hampered by the lack of suitable animal models (10,30). Thus, the few models that have been used previously, for example, rabbit ileal loops or the rabbit intestine nonligated (RITARD) model (10,30), are very laborious and may not accurately allow evaluation of protective immunity in the small intestine, with the exception of toxin immunity induced by patches where protective effects in animals have predicted field efficacy (42). Hence, it has been identified as a priority in ETEC vaccine research to develop a simple animal model allowing screening of both live and inactivated vaccine candidates. There are also urgent needs to define immunological markers of protection against candidate ETEC vaccines in humans. This includes identification of simple and appropriate immunological methods that may reflect intestinal immune responses against mucosal vaccines, with a focus on young children and infants, in different vaccine trials (30,31). Work is also in progress in adult human volunteers to try to identify markers of protection after immunization with live ETEC and protection against rechallenge with fully virulent ETEC bacteria. Studies have also been initiated to identify immune responses that may be associated with protection against reinfection with ETEC in children in high endemic areas (3). The ultimate determination of protective efficacy and correlates of protection will, however, have to rely on results from phase III studies in children in endemic areas and in travelers from industrialized countries to ETEC-endemic countries. Such studies may be preceded by evaluations of candidate vaccines for protective efficacy in challenged volunteers. However, it remains to be seen how well results from challenge studies in adults in nonendemic countries and field trials in travelers will predict the efficacy of the same ETEC vaccines in infants and young children in developing countries.

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Chapter 65: Vaccines Against Enterotoxigenic E. coli 23. Wennera˚s C, Qadri F, Bardhan PK, et al. Intestinal immune responses in patients infected with enterotoxigenic Escherichia coli and in vaccinees. Infect Immun 1999; 67:6234–6241. 24. Qadri F, Ahmed T, Ahmed F, et al. Mucosal and systemic immune responses in patients with diarrhea due to CS6-expressing enterotoxigenic Escherichia coli. Infect Immun 2007; 75:2269–2274. 25. Qadri F, Ahmed F, Ahmed T, et al. Homologous and crossreactive immune responses to enterotoxigenic Escherichia coli colonization factors in Bangladeshi children. Infect Immun 2006; 74:4512–4518. 26. Peltola H, Siitonen A, Kyronseppa H, et al. Prevention of travellers’ diarrhea by oral B-subunit/whole-cell cholera vaccine. Lancet 1991; 338:1285–1289. 27. Frech SA, Kenney RT, Spyr CA, et al. Improved immune responses to influenza vaccination in the elderly using an immunostimulant patch. Vaccine 2005; 23:946–950. 28. Steinsland H, Valentiner-Branth P, Gjessing HK, et al. Protection from natural infections with enterotoxigenic Escherichia coli: longitudinal study. Lancet 2003; 362:286–291. 29. Lo´pez-Gigosos R, Garcı´a-Fortea P, Reina-Don˜a E, et al. Effectiveness in prevention of travellers’ diarrhea by an oral cholera vaccine WC/rBS. Travel Med Infect Dis 2007; 5:380–384. 30. Walker RI, Steele D, Aguado T. Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 2007; 25:2545–2566. 31. WHO Weekly Epidemiological Record, No. 10, 2008:92–95. 32. Lebens M, Shahabi V, Ba¨ckstro¨m M, et al. Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad spectrum vaccine. Infect Immun 1996; 64:2144–2150. 33. Deen JL, Von Seidlein L, Sur D, et al. The high burden of cholera in children: comparison of incidence from endemic areas in Asia and Africa. PLoS Negl Trop Dis 2008; 2:e173. 34. Gu¨erenˇa-Burguenˇo F, Hall ER, Taylor DN, et al. Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously. Infect Immun 2002; 70:1874–1880. 35. Glenn GM, Villar CP, Flyer DC, et al. Safety and immunogenicity of an enterotoxigenic Escherichia coli vaccine patch containing heat-labile toxin: use of skin pretreatment to disrupt the stratum corneum. Infect Immun 2007; 75:2163–2170. 36. McKenzie R, Bourgeois AL, Frech SA, et al. Transcutaneous immunization with the heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC): protective efficacy in a double-blind, placebo-controlled challenge study. Vaccine 2007; 25:3684–3691. 37. Frech SA, Dupont HL, Bourgeois AL, et al. Use of a patch containing heat-labile toxin from Escherichia coli against travellers’ diarrhea: a phase II, randomised, double blind, placebo controlled field trial. Lancet 2008; 371:2019–2025. 38. Frerichs DM, Ellingsworth LR, Frech S, et al. Controlled, singlestep, stratum corneum disruption as a pretreatment for immunization via a patch. Vaccine 2008; 26:2782–2787. 39. Johnson AM, Kaushik RS, Francis DH, et al. Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells. J Bacteriol 2009; 191:178–185. 40. Look JL, Butler B, Al-Khalili M, et al. The adjuvant patch: a universal dose sparing approach for pandemic and conventional vaccines. BioPharm Int Guide to Vaccine Dev Manuf 2007; 20(suppl):34–45. 41. Jobling M, Rasulova F, Poole St, et al. Optimized production and evaluation of pilus adhesin plus enterotoxin B subunit chimers as novel candidate vaccines against diarrhea caused by enterotoxigenic E. coli. Cholerae & other bacterial enteric infections. United States-Japan Cooperative Medical Science program. 42nd Annual Joint Panel Meeting, Austin, Texas, December 5–7, 2007. 42. Yu J, Cassels F, Sharton-Kersten T, et al. Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli. Infect Immun 2002; 70:756–768.

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43. Katz DE, DeLorimier AJ, Wolf MK, et al. Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen. Vaccine 2003; 21:341–346. ˚ hre´n C, Jertborn M, Svennerholm A-M. Intestinal immune 44. A responses to an inactivated oral enterotoxigenic Escherichia coli vaccine and associated immunoglobulin A responses in blood. Infect Immun 1998; 66:3311–3316. ˚ hre´n C, Svennerholm A-M. Dose-dependent circu45. Jertborn M, A lating immunoglobulin A antibody-secreting cell and serum antibody responses in Swedish volunteers to an oral inactivated enterotoxigenic Escherichia coli vaccine. Clin Diagn Lab Immunol 2001; 8:424–428. 46. Savarino SJ, Brown FM, Hall E, et al. Safety and immunogenicity of an oral, killed enterotoxigenic Escherichia coli-cholera toxin B subunit vaccine in Egyptian adults. J Infect Dis 1998; 177: 796–799. 47. Savarino SJ, Hall ER, Bassily S, et al. Introductory evaluation of an oral, killed whole cell enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Egyptian infants. Pediatr Infect Dis J 2002; 21:322–330. 48. Qadri F, Ahmed T, Ahmed F, et al. Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi children 18–36 months of age. Vaccine 2003; 21:2394–2403. 49. Qadri F, Ahmed T, Ahmed F, et al. Reduced doses of oral killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine is safe and immunogenic in Bangladeshi infants 6–17 months of age: dosing studies in different age groups. Vaccine 2006; 24:1726–1733. 50. Wiedermann G, Kollaritsch H, Kundi M, et al. Double-blind, randomized, placebo controlled pilot study evaluating efficacy and reactogenicity of an oral ETEC B-subunit-inactivated whole cell vaccine against travelers’ diarrhea. J Travel Med 2000; 7: 27–29. 51. Sack DA, Shimko J, Torres O, et al. Randomised, double-blind, safety and efficacy of a killed oral vaccine for enterotoxigenic E. coli diarrhea of travellers to Guatemala and Mexico. Vaccine 2007; 25:4392–4400. 52. Bourgeois AL, Halpern J, Grahek S, et al. Vaccination of travellers to Guatemala (GU) and Mexico (MX) with an oral killed vaccine (OKV) for enterotoxigenic E coli (ETEC): impact of vaccine ‘‘take’’ on risk of ETEC disease and infection with enteric pathogens. 4th International Conference on Vaccines for Enteric Diseases (VED 2007). Lisbon, Portugal, April 25–27, 2007. 53. Grimwood K, Bines JE. Rotavirus vaccines must perform in lowincome countries too. Lancet 2007; 370:1739–1740. 54. Tobias J, Lebens M, Bo¨lin I, et al. Construction of non-toxic Escherichia coli and Vibrio cholerae strains expressing high and immunogenic levels of enterotoxigenic E. coli colonization factor I fimbriae. Vaccine 2008; 26:743–752. 55. Tobias J, Lebens M, Ka¨llga˚rd S, et al. Role of different genes in the CS6 operon for surface expression of enterotoxigenic Escherichia coli colonization factor CS6. Vaccine 2008; 26:5373–5380. 56. Freytag LC, Clements JD. Mucosal adjuvants. Vaccine 2005; 23:1804–1813. 57. Clements J. LT adjuvants. 4th International Conference on Vaccines for Enteric Diseases (VED 2007). Lisbon, Portugal, April 25–27, 2007. 58. Cuburu N, Kweon MN, Song JH, et al. Sublingual immunization induces broad-based systemic and mucosal immune responses in mice. Vaccine 2007; 25:8598–8610. 59. Turner AK, Terry TD, Sack DA, et al. Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans. Infect Immun 2001; 69:4969–4979. 60. McKenzie R, Bourgeois AL, Engstrom F, et al. Comparative safety and immunogenicity of two attenuated enterotoxigenic Escherichia coli vaccine strains in healthy adults. Infect Immun 2006; 74: 994–1000.

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66 Multivalent Shigella Enterotoxigenic Escherichia coli Vaccine Eileen M. Barry and Myron M. Levine Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION Enterotoxigenic Escherichia coli and Shigella are important causes of diarrheal disease in infants and young children in developing countries and are major etiological agents of traveler’s diarrhea (1–8).

SHIGELLA Strategy to Achieve Broad-Spectrum Protection Against Shigella A globally useful Shigella vaccine will have to protect against Shigella dysenteriae 1 (cause of severe epidemic disease in the least developed countries), all 14 serotypes of Shigella flexneri (main cause of endemic shigellosis in developing countries), and Shigella sonnei (the serotype most frequently associated with traveler’s shigellosis and the most common serotype causing disease in industrialized countries). Infection-derived immunity against Shigella is directed toward O antigens (3). On the basis of shared O antigens among the S. flexneri serotypes (other than S. flexneri 6), Noriega et al. (9) hypothesized that a vaccine containing S. flexneri 2a, S. flexneri 3a, and S. flexneri 6 O antigens could provide broad protection against all 14 S. flexneri serotypes (Tables 1 and 2). With the exception of S. flexneri 6, the O antigenic structure of all the other S. flexneri serotypes and subtypes consists of tetrasaccharide (rhamnose-rhamnoserhamnose-N-acetylglucosamine) repeat backbone (3). Linkage of D-glucose or O-acetyl moieties at various sites on the backbone results in distinct type and group O antigens. The group antigens are shared among different S. flexneri types, other than S. flexneri 6 (3). By means of challenge studies in guinea pigs, Noriega et al. (9) generated preclinical evidence supporting the hypothesis of cross-protection among certain S. flexneri serotypes on the basis of shared group antigens. Pursuing this strategy, investigators at the Center for Vaccine Development have concluded that a multivalent Shigella vaccine that includes O antigens of five carefully selected serotypes, S. dysenteriae 1, S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei, could provide broad coverage against the most important Shigella serotypes that cause disease worldwide (1–3,9) (Tables 1 and 2).

Shigella Harboring Mutations in guaBA and in the Genes Encoding Shigella Enterotoxins 1 and 2 Introduction of a deletion mutation in the guaBA operon (which impairs guanine nucleotide biosynthesis) of S. flexneri 2a wild-

type strain 2457T renders strain CVD 1204, which is markedly attenuated compared with its wild-type parent (10). Nevertheless, at high dosage levels in humans, approximately one half of subjects who ingested CVD 1204 still developed mild diarrhea (10) Full clinical attenuation was not achieved until deletions in the genes encoding Shigella enterotoxin (ShET)1 (set) (11,12) and ShET2 (sen) (13) were also introduced, resulting in the welltolerated and immunogenic strain CVD 1208 (10). On the basis of theoretical concerns for the presence of adventitious agents that might be transferred from bacterial growth media to the vaccine strains, CVD 1208S was fully reconstructed from the original wild-type strain using soybased media, which was completely free of animal components. CVD 1208S was similarly well tolerated and immunogenic in volunteers as CVD 1208 (14). On the basis of the favorable experience with S. flexneri 2a as a prototype, attenuated S. dysenteriae 1, S. flexneri 3a, S. flexneri 6, and S. sonnei strains are also being constructed (2,9) that similarly harbor deletions in guaBA and sen (Table 1); in addition, the S. dysenteriae 1 strain has a deletion mutation in stxA, which encodes the A subunit of Shiga toxin. Collectively, these five carefully selected Shigella serotypes should provide broad coverage against the most important serotypes that cause shigellosis worldwide (9).

ENTEROTOXIGENIC ESCHERICHIA COLI Target Antigens As a preliminary step in the pathogenesis of diarrhea, enterotoxigenic eschericiha coli (ETEC) adhere to receptors on enterocytes in the proximal small intestine by means of fimbrial colonization factors, thereby counteracting the persistalsis defense mechanism (2). Once adherent, they elaborate enterotoxins that cause intestinal secretion, culminating clinically in diarrhea. Considerable evidence, as reviewed in chapter 65, indicates that broad-spectrum, relatively long-lived immunity to ETEC is mediated by intestinal immune responses directed against these fimbrial attachment factors (16).

Antigenic Diversity Among Human ETEC Pathogens Analysis of the antigenic structure of enterotoxigenic E. coli strains from endemic areas shows many different O:H serotypes, at least ten distinct antigenic types of fimbrial colonization factors

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Table 1 Components of the CVD Multivalent Shigella/ETEC Combination Live Vector Vaccine Shigella serotype

Strain designation

Attenuating mutations

ETEC fimbriae or heat-labile enterotoxin antigen expressed

S. S. S. S. S.

CVD CVD CVD CVD CVD

guaBA, guaBA, guaBA, guaBA, guaBA,

CS4, CS6 Colonization factor antigen/I, LTh B subunit CS1, CS5 LTh B subunit and/or CS17 CS2, CS3

dysenteriae 1 flexneri 2a flexneri 3a flexneri 6 sonnei

1256 1208S 1213S 1215S 1233S

stx, sen set, sen sen sen sen

Abbreviations: CS, coli surface antigen; LTh, heat-labile enterotoxin from human ETEC pathogens.

Table 2 Shigella flexneri Antigens Expressed by the Three Serotypes (S. flexneri 2a, S. flexneri 3a, and S. flexneri 6) in the CVD Shigella/ETEC Combination Vaccine and Cross-Reactivity with Other S. flexneri Serotypes Shigella serotype S. flexneri 2a S. flexneri 3a S. flexneri 6

Type antigen II – VI

Group antigens 3, 4 6 and 7, 8 –

[of which the most common are colonization factor antigen (CFA)/I and coli surface antigen (CS)1-CS6], and three different toxin phenotypes heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), and LT/ST (4,16). CFA/I is a single antigenic moiety. CS1, CS2, and CS3 constitute the CFA/II family of antigens. All CFA/II strains express CS3, either alone or in conjunction with CS1 or CS2. CS4, CS5, and CS6 comprise the CFA/IV family of antigens. All CFA/IV strains express CS6, either alone or in conjunction with CS4 or CS5. Other fimbrial colonization factors are much less frequent. Carriage of the genes that encode a particular fimbrial colonization factor is closely correlated with O:H serotype and toxin phenotype. Analysis of ETEC isolates from diverse geographic areas shows that CFA/I and CS1-6 are found on the majority of isolates. Analysis of strain collections from patients with traveler’s diarrhea or endemic pediatric diarrhea shows that ca. 70% to 90% of isolates that elaborate both LT and ST express these CFAs, while they are found on ca. 60% of ST-only strains. Generally, less than 10% of LT-only strains bear these CFAs. Thus, a multivalent ETEC vaccine that contained CFA/I and CS1-6 plus an appropriate antigen (such as B subunit or mutant LT) to elicit neutralizing LT antitoxin might broaden protection to cover approximately 80% to 90% of ETEC strains worldwide. Inclusion of less frequent fimbrial antigens in a multivalent vaccine could expand the spectrum of coverage, albeit at the price of even greater complexity.

Infection-Derived Immunity to ETEC Despite the antigenic heterogeneity of ETEC, evidence from both volunteer studies and epidemiological surveys argues convincingly that prior clinical infection with enterotoxigenic E. coli confers immunity (5,6,17). In endemic areas, multiple infections with strains bearing different fimbrial colonization factors must occur for broad-spectrum immunity to be elicited. In less developed countries, infants and young children experience up to three separate clinical ETEC infections per year during the first three years of life, after which the incidence of ETEC diarrhea plummets (5). The lower incidence in older persons is due to specific acquired immunity rather than to nonspecific age-related host factors, since adult travelers from industrialized countries who visit less developed countries where ETEC pediatric diarrhea is endemic suffer high attack rates of ETEC travelers’ diarrhea. The fimbrial colonization factor antigenic profiles of strains that cause endemic pediatric

Shigella flexneri serotypes that cross-react 2b, 1a, 3b, 4a, 5a, Y 3b, 1b, 2b, 4b, 4c, 5b, X –

diarrhea in developing countries and those that cause travelers’ diarrhea are the same. Travelers from industrialized countries who remain in less developed countries for at least a year and travelers who arrive from other less developed countries suffer significantly lower incidence rates of ETEC diarrhea than newly arrived travelers from industrialized countries (6). These data further support the concept of acquired immunity. Data from a prospective epidemiological field study in Mexican infants and young children provide direct evidence that acquired immunity is largely directed at fimbrial colonization factors of ETEC (18).

Lessons Learned from Studies with a Prototype Attenuated Escherichia coli Live Oral ETEC Vaccine E. coli E1392-75-2A is a CFA/II-positive mutant strain derived in the Central Public Health Laboratory, Colindale, U.K. wherein the genes encoding LT and ST spontaneously deleted from the CFA/II plasmid. Consequently, E1392-75-2A, which expresses CS1 and CS3 fimbrial antigens, is negative when tested with toxin assays and gene probes for LT and ST. Levine et al. (7,19,20) utilized strain E1392-75-2A to explore fundamental questions of anticolonization immunity in the absence of antitoxic immunity. All volunteers who were fed 1010 CFU of strain E1392-75-2A developed significant rises in intestinal fluid SIgA antibody to CS1 and CS3 fimbriae. The geometric mean titer (GMT) of anti-fimbrial CS1 and CS3 SIgA antibody in these volunteers was 10-fold higher than the peak postvaccination GMT of volunteers who received enteral immunization with multiple doses of purified CS1 and CS3 fimbriae. A group of vaccinees who were immunized with a single 5  1010 CFU dose of E1392-75-2A with buffer were challenged one month later, along with unimmunized control volunteers. The pathogenic ETEC challenge strain used, E24377A, was of an heterologous serotype O139:H28 but expressed CS1 and CS3 and elaborated LT and ST. The vaccinees were significantly protected (p < 0.005, 75% vaccine efficacy) against ETEC diarrhea (7). Bacteriological studies showed that anticolonization immunity was responsible for the protection. In the challenge study, all participants, both vaccinees and unimmunized controls, excreted the ETEC challenge strain, and there was no difference between the groups in the mean number of ETEC per gram of stool. In contrast, a striking difference was found in

Chapter 66: Multivalent Shigella Enterotoxigenic E. coli Vaccine

duodenal cultures that monitored colonization of the proximal small intestine, the critical site of ETEC-host interaction. The challenge strain was recovered from duodenal cultures of five of six controls (mean 7  103 CFU/mL) versus only 1 of 12 vaccinees (101 CFU/mL) (p < 0.004). Levine et al. interpreted these results to mean that SIgA anti-CS1 and anti-CS3 fimbrial antibody in the proximal intestine stimulated by the live oral vaccine prevented challenge ETEC from colonizing the proximal small intestine. Since the immune response was not bactericidal, the ETEC organisms were carried by peristalsis to the large intestine where they could colonize without causing diarrheal illness. Strain E1392-75-2A caused mild diarrhea in approximately 15% of the recipients who ingested it, an unacceptable rate of adverse reactions that made it unworthy of further development. Nevertheless, this strain provided invaluable data on the feasibility of eliciting protection in humans mediated by immune responses directed against fimbrial colonization factors.

MULTIVALENT SHIGELLA-ETEC LIVE VECTOR VACCINE Attenuated Shigella Strains Expressing ETEC Antigens Investigators at the Center for Vaccine Development of the University of Maryland have shown that attenuated Shigella can be used as live vector vaccines to express ETEC fimbrial antigens (Fig. 1) and LT toxoids (B subunit or mutant LT) and deliver them to the immune system, resulting in SIgA anti-fimbrial and anti-LT responses (21–26), as well as Shigella anti-O antibody responses in mucosal secretions and serum.

The Multivalent Vaccine The multivalent Shigella/ETEC vaccine under development contains five attenuated Shigella serotype strains (S. dysenteriae 1, S. flexneri 2a, S. flexneri 3a, and S. sonnei), each expressing two different ETEC fimbrial antigens and an antigen (‘‘LT toxoid’’) to stimulate neutralizing LT antitoxin (Table 1). Both LT B subunit and mutant LT moieties are being evaluated as possible LT toxoids, the former having the attraction of greater potential safety. Heat-labile enterotoxin from human ETEC pathogens (LTh), from domestic animals, and cholera toxin, or their respective B subunits, can all elicit antibodies that can neutralize LTh

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(27). However, each of these antigens has unique epitopes, and the highest antitoxin titers observed are against the homologous antigen (27–29). Consequently, it is our contention that an antigen based on LTh should be used to stimulate LT antitoxin. Notably, expression of the ETEC fimbriae and LT toxoid does not diminish the capacity of the vector strain to protect against challenge with wild-type Shigella in a guinea pig model (21–26).

Shigella Can Express Combinations of ETEC Fimbria Not Found in Nature The utility of attenuated Shigella as live vectors to coexpress CFA/I and CS3 fimbriae of ETEC, a combination never found in nature, was documented (21). The immunogenicity of this bivalent Shigella live vector strain was evaluated in a guinea pig immunization model in which Shigella are administered intranasally following which mucosal SIgA responses are measured in tears. The Shigella live vector expressing these two ETEC fimbrial antigens elicited SIgA mucosal antibody responses to both CFA/I and CS3 (21,26).

Genetic Characterization of CS4 In constructing the multivalent Shigella live vector ETEC vaccine, one must be able to manipulate the operons encoding biogenesis of each of the fimbrial structures in the vaccine (CFA/I and CS1-CS6). Among these, the CS4 operon was the last to be elucidated and cloned (24). Altboum et al. (24) showed that the CS4 (csa) operon encodes a 17-kDa major fimbrial subunit (CsaB), a 40-kDa fimbrial tip protein (CsaE), a 27-kDa chaperone (CsaA), and a 97-kDa usher protein (CsaC). Furthermore, they showed that the predicted amino acid sequences of CS4 proteins are highly homologous to CS1, CS2, and CFA/I. With the successful cloning and expression of CS4, it is now possible to express all of the common ETEC fimbrial colonization factors in Shigella live vectors. When CS4 was expressed in the attenuated Shigella live vector and used to immunize guinea pigs, robust serum and mucosal responses were elicited against CS4 (24).

Combinations of Shigella Live Vector Strains Expressing Different ETEC Fimbriae A hurdle faced by a multivalent live vector Shigella/ETEC vaccine is to demonstrate convincingly (ultimately in humans)

Figure 1 Expression of CFA/I fimbriae on Shigella flexneri 2a vaccine strain CVD 1204. Electron microscopy of negatively stained S. flexneri 2a strain CVD 1204 (A) and CVD 1204 (pCFA/I) (B). Many fimbriae displaying typical CFA/I morphology are visible in (B) (arrows). Abbreviation: CFA, colonization factor antigen.

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that a mixture of vaccine strains, each expressing different ETEC fimbrial antigens, can elicit strong mucosal responses to all the different antigens in the combination. Toward this goal, initial preclinical studies in the guinea pig model were performed with two bivalent Shigella live vector candidates, one expressing CFA/I and CS3 (21) and the other CS2 and CS3 (23). Each bivalent live vector elicited strong mucosal SIgA responses in tears against both fimbrial antigens; serum antibody responses were also robust. Studies were next undertaken with a pentavalent Shigella/ETEC vaccine consisting of attenuated S. flexneri 2a expressing either CFA/I, CS2, CS3, CS4 fimbriae or mutant LTh (20). Following intranasal immunization, SIgA anti-fimbrial responses were measured in tears of guinea pigs immunized with either one of the monovalent vaccines or the multivalent vaccine (containing the five different live vector strains administered in combination). For each of the ETEC antigens, the combination vaccine, as well as each corresponding monovalent vaccine, stimulated significant rises in anti-fimbrial or anti-LT antibody. These groups of guinea pigs also exhibited strong Shigella anti-O antibody responses and were protected against challenge with wild-type S. flexneri 2a (20). Finally, a combination of three different live attenuated strains each expressing one or two ETEC antigens was used to assess the potential of a multivalent vaccine to elicit responses against all components. A mixture composed of S. flexneri 2a strain CVD 1208 expressing CFA/I and CS3 plus S. sonnei strain CVD 1233 expressing CS4 and LThK63 plus S. dysenteriae 1 strain CVD 1252 expressing CS2 was able to elicit serum and mucosal antibody responses against each ETEC antigen and each Shigella strain included. Responses were not diminished in the animals inoculated with the trivalent combination compared with each individual strain alone. Furthermore, the immunized animals were protected against challenge with the wild-type version of each Shigella strain (26).

Shigella Live Vector Strains Expressing ETEC Fimbriae and LT Toxoids Plasmids have been constructed that carry operons both for fimbrial biogenesis and for either mutant LTh or LT B subunit expression so that both anti-fimbrial and antitoxin responses can be stimulated (22,25). Preclinical studies with these constructs, individually and in combination, evaluated these different strategies and paved the way for proof-of-principle clinical trials. The first prototype strain, CVD 1208S(pCFA/ I-LThA2B), has entered phase I clinical trials. Resultant safety and immunogenicity data will guide advancement and further development of this multivalent vaccine strategy. In a related strategy, attenuated Shigella strains harboring LT toxoid genes integrated into the chromosome (and under control of a variety of promoters) and carrying plasmids allowing expression of ETEC fimbriae have been constructed. Yet another strategy involves the expression of both CFA fimbriae and LT B subunit from genes integrated into chromosomal loci.

SUMMARY COMMENT A multivalent Shigella/ETEC vaccine is complex in development and with respect to the control of manufactured lots. On the other hand, it has a number of distinct potential advantages. A single, albeit complex, vaccine would offer broad-spectrum coverage against two pathogens for which the target populations are the same, travelers and infants in developing countries.

REFERENCES 1. Kotloff KL, Winickoff JP, Ivanoff B, et al. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ 1999; 77(8): 651–666. 2. Levine MM. Immunization against bacterial diseases of the intestine. J Pediatr Gastroenterol Nutr 2000; 31(4):336–355. 3. Levine MM, Kotloff KL, Barry EM, et al. Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road. Nat Rev Microbiol 2007; 5(7):540–553. 4. Levine MM, Ferreccio C, Prado V, et al. Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile. Am J Epidemiol 1993; 138(10):849–869. 5. Black RE, Merson MH, Rowe B, et al. Enterotoxigenic Escherichia coli diarrhoea: acquired immunity and transmission in an endemic area. Bull World Health Organ 1981; 59(2):263–268. 6. DuPont HL, Olarte J, Evans DG, et al. Comparative susceptibility of latin american and united states students to enteric pathogens. N Engl J Med 1976; 295(27):1520–1521. 7. Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis 1987; 155(3):377–389. 8. Sanders JW, Frenck RW, Putnam SD, et al. Azithromycin and loperamide are comparable to levofloxacin and loperamide for the treatment of traveler’s diarrhea in United States military personnel in Turkey. Clin Infect Dis 2007; 45(3):294–301. 9. Noriega FR, Liao FM, Maneval DR, et al. Strategy for crossprotection among Shigella flexneri serotypes. Infect Immun 1999; 67(2):782–788. 10. Noriega FR, Losonsky G, Lauderbaugh C, et al. Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine. Infect Immun 1996; 64(8):3055–3061. 11. Fasano A, Noriega FR, Maneval DR Jr., et al. Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro. J Clin Invest 1995; 95(6):2853–2861. 12. Fasano A, Noriega FR, Liao FM, et al. Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut 1997; 40(4): 505–511. 13. Nataro JP, Seriwatana J, Fasano A, et al. Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains. Infect Immun 1995; 63(12):4721–4728. 14. Kotloff KL, Simon JK, Pasetti MF, et al. Safety and immunogenicity of CVD 1208S, a live, oral deltaguaBA deltasen deltaset Shigella flexneri 2a vaccine grown on animal-free media. Hum Vaccin 2007; 3(6):268–275. 15. Walker RI, Steele D, Aguado T. Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 2007; 25(14):2545–2566. 16. Levine MM, Giron JA, Noriega F. Fimbrial vaccines. In: Klemm P, ed. Fimbriae: Adhesion, Biogenics, Genetics, and Vaccines. Boca Raton: CRC Press, 1994:255–270. 17. Levine MM, Nalin DR, Hoover DL, et al. Immunity to enterotoxigenic Escherichia coli. Infect Immun 1979; 23(3):729–736. 18. Cravioto A, Reyes RE, Ortega R, et al. Prospective study of diarrhoeal disease in a cohort of rural Mexican children: incidence and isolated pathogens during the first two years of life. Epidemiol Infect 1988; 101(1):123–134. 19. Levine MM. Travellers’ diarrhoea: prospects for successful immunoprophylaxis. Scand J Gastroenterol Suppl 1983; 84:121–134. 20. Levine MM, Black RE, Clements ML, et al. Prevention of enterotoxigenic E. coli diarrheal infectionby vaccines that stimulate antiadhesion (antipili) immunity. In: Boedeker EC, ed. Attachment of Organisms to the Gut Mucosa. Boca Raton: CRC Press, 1984:223–244. 21. Noriega FR, Losonsky G, Wang JY, et al. Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for

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delivering antigens of enterotoxigenic Escherichia coli. Infect Immun 1996; 64(1):23–27. Koprowski H, Levine MM, Anderson RJ, et al. Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli. Infect Immun 2000; 68(9):4884–4892. Altboum Z, Barry EM, Losonsky G, et al. Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection. Infect Immun 2001; 69(5):3150–3158. Altboum Z, Levine MM, Galen JE, et al. Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain. Infect Immun 2003; 71(3):1352–1360. Barry EM, Altboum Z, Losonsky G, et al. Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae

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67 Vaccines for Staphylococcus aureus Infections Juliane Bubeck Wardenburg Departments of Pediatrics and Microbiology, University of Chicago, Chicago, Illinois, U.S.A.

Dominique Missiakas and Olaf Schneewind Department of Microbiology, University of Chicago, Chicago, Illinois, U.S.A.

INTRODUCTION Nearly three quarters of a century after the introduction of the potent anti-staphylococcal drug penicillin, Staphylococcus aureus remains a significant bacterial cause of morbidity and mortality in the human population. This gram-positive organism exists as a commensal in the human, residing in the nares or on the skin of approximately one-third of individuals at any time (1,2). The epithelial layer of the skin and mucous membranes proves to be a potent host defense mechanism against staphylococcal infection. A breach of this barrier, however, predisposes the host to a myriad of disease manifestations resulting from S. aureus invasion of the tissues. The remarkable pathogenic potential of this organism has been demonstrated over the past decade, with the rapid spread of highly virulent S. aureus strains worldwide (3–6). A collection of features distinguishes these strains from those previously associated with disease. Most isolates carry the SCCmec IV genetic element that confers resistance to b-lactam antimicrobials, rendering this entire class of antimicrobials obsolete (7). In addition, they demonstrate a novel epidemiologic pattern, frequently being transmitted outside the hospital environment, among otherwise healthy individuals; thus they have been designated community-associated methicillin-resistant S. aureus (CA-MRSA) (8–10). Finally, a growing number of studies have defined unique virulence traits expressed by these strains. The factor that has garnered the majority of attention by virtue of its high degree of epidemiologic association with invasive S. aureus disease is Panton-Valentine leukocidin (PVL), a pore-forming cytotoxin with specificity for leukocytes (11–15). The genes encoding PVL are present on a bacteriophage, a mobile genetic element that contributes to genomic plasticity through horizontal gene transfer (16). Additional phage-encoded proteins that have been demonstrated to contribute to the virulence phenotype include the plasminogen activator staphylokinase (Sak) (17–19), the immunomodulatory proteins CHIPS (chemotaxis inhibiting protein) and SCIN (staphylococcal complement inhibitor) (20–22). Most recently, Wang et al., have defined a novel class of secreted staphylococcal peptides termed ‘‘phenol-soluble modulins’’ (PSMs) that are highly expressed in current CA-MRSA isolates, and contribute to the destruction of human neutrophils (23). While it is unlikely that a single factor in CA-MRSA strains is solely responsible for the high virulence phenotype, it is readily appreciated that a constellation of pathogenic traits may render these strains more capable of causing significant infection in healthy hosts.

The emergence of these strains, coupled with current antimicrobial resistance patterns, has raised concern of the potential of this pathogen to reach epidemic proportions (24,25). The cumulative burden of S. aureus infection heightens the demand for vaccines that are capable of inducing protection against a wide array of disease manifestations within a broad population of individuals. This approach clearly necessitates the targeting of bacterial virulence factors that are essential to the pathogenesis of the organism, irrespective of the specific type of infection. Further, the current spectrum of disease observed in the pre-vaccine era requires novel strategies to facilitate early identification of the pathogen and the development of diseasespecific immunotherapy to be used independently or in concert with antimicrobial drugs.

PATHOGENESIS OF STAPHYLOCOCCUS AUREUS INFECTION S. aureus achieves success as a pathogen through a combination of factors. First, its close relationship with the human host as a commensal positions the organism in immediate proximity to the tissues in which it is suited to cause disease. Indeed, colonization with S. aureus is a significant risk factor for the development of invasive disease (26–28). Second, the dynamic spread of the organism is facilitated primarily through personto-person contact. The human population thereby serves as a ready conduit for transmission. Lastly, a number of virulence factors intrinsic to the organism work together in a concerted fashion to permit host tissue invasion, bacterial proliferation, and evasion of the host defense, culminating in the spread of the pathogen.

Clinical Manifestations of Disease Essentially, every organ system and tissue of the human is susceptible to infection with S. aureus. The most common site of infection is the skin and soft tissues, however this pathogen also results in frequent infection of the deep tissues, causing pneumonia upon replication in the lungs, osteomyelitis of the skeletal system, and endocarditis when affecting the lining of the heart (29). Bloodstream infection, or septicemia, is often related to seeding of these deeper organs, and in and of itself accounts for approximately 75,000 cases of disease per year in the United States alone (30). The direct consequence of S. aureus infection of specific tissues is further confounded by a number

Chapter 67: Vaccines for S. aureus Infections

of toxin-mediated syndromes that are also capable of causing significant morbidity and mortality. The most well known among these is staphylococcal toxic shock syndrome, a systemic inflammatory disease mediated by toxic shock syndrome toxin (TSST)-induced polyclonal T-cell activation (29,31,32). A large collection of staphylococcal enterotoxins contributes to disease of the gastrointestinal system (33), while a family of epidermolytic toxins can cause life-threatening desquamation (34). This wide array of disease processes is typical of S. aureus; the advent of CA-MRSA strains has led to the observation of several newer manifestations of disease including severe sepsis, necrotizing pneumonia, and necrotizing fasciitis (15,35–39). Together, these infections impact on millions within the human population, and place a substantial burden on the health care system.

Epidemiology of Staphylococcus aureus Infection Historically, significant S. aureus infections were most often associated with the hospital environment. Staphylococci, as a part of the skin flora, capitalize on the disruption of the epithelium that is commonplace in the hospital environment. In-dwelling catheters, medical devices, and surgical wounds are commonly infected by S. aureus. Debilitated patients, those with chronic underlying disease and those receiving intensive care therapies are most at risk for the development of nosocomial infection with S. aureus. Drug resistance amongst S. aureus was first appreciated in the hospital environment. Within six years following the introduction of penicillin in 1941, a 25% resistance rate was reported for S. aureus isolates; this was followed by the introduction of the semi-synthetic methicillin in 1961, with MRSA phenotypes emerging shortly thereafter (10). An assessment of methicillin resistance among S. aureus isolates in intensive care units in 2003 determined that over 60% of hospital-acquired strains are no longer susceptible to this class of drugs (40). A study conducted in late 2006 by the Association for Professionals in Infection and Epidemiology demonstrated the prevalence of MRSA strains in hospitalized individuals to be 46 per 1000, approximately 10-fold greater than previously estimated (41). The initial observation of CA-MRSA in healthy adults and children heralded the widespread public health threat that now exists. Strains responsible for community-associated disease are most often resistant to b-lactams but maintain susceptibility to other antimicrobials. A recent study by Klevens et al., assessing the incidence of MSRA disease within nine communities in the United States documented that nearly 14% of all invasive MRSA disease originates in the community (42). In total, the authors estimated that greater than 94,000 cases of invasive MRSA occurred in the United States alone in 2005, resulting in over 18,000 deaths (42).

Host Defense Against Staphylococcus aureus Infection The principal defense against S. aureus infection resides in the neutrophil. The most compelling genetic demonstration of the role of the neutrophil in protection against S. aureus infection is seen in patients suffering from chronic granulomatous disease (CGD), a genetic disorder that renders the patient’s neutrophils incapable of generating a cellular oxidative response. The molecular basis for this disease is a mutation in the multisubunit NADPH oxidase complex responsible for the generation of the superoxide radical in the phagocytic vacuole (43).

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Reactive oxygen species, along with the acidic vacuolar environment, prove toxic to S. aureus, and serve as a primary early means by which to curtail bacterial spread. Patients afflicted with CGD suffer from recurrent S. aureus infection. Several additional components of the innate immune system enhance the early host response to S. aureus infection. Antimicrobial peptides (AMPs) such as defensins and cathelicidins are present on mucosal and epithelial surfaces, and facilitate direct lysis of the invading pathogen (44,45). Mutation of S. aureus genes encoding the components of the AMP sensor system compromises the ability of the pathogen to survive during murine infection (46). Proteins of the complement cascade are also important in innate host defense against S. aureus (47–49). These proteins serve a twofold role—first, several components are capable of binding to the staphylococcal surface, thereby facilitating phagocytic uptake of the pathogen. Second, proteolytic fragments of C3 and C5 are potent chemoattractant peptides for phagocytes, serving to amplify the host response. Underscoring the importance of complement in staphylococcal clearance, complement depletion in experimental animals renders them more susceptible to septicemia (50). The role of the adaptive immune system in protection against S. aureus has not been well elucidated. B cell function clearly facilitates the generation of antibodies specific for S. aureus, as these are both present in humans and are known to rise following infection (51,52). Anti-staphylococcal antibodies likely serve the dual role of neutralizing staphylococcal exotoxins and enhancing the phagocytic uptake of staphylococci. It is well appreciated that the generation of specific antibody responses against protein antigens requires a cognate T-cell response to the pathogen. However, the precise role of T cells in anti-staphylococcal immunity is not yet well defined. In fact, studies using surgical wound site infection in an animal model of disease suggest that T-cell recruitment may enhance abscess formation, leading to a localized accumulation of bacteria (53). Mechanistically, the recruited T cells appear to secrete chemokines of the CXC family, thereby augmenting the recruitment of phagocytes to the site of infection (53). It is clear from these data that the host immune system exerts a multifaceted attack on S. aureus, requiring a complex response on the part of the pathogen to evade these defenses.

Staphylococcal Virulence Programs The staphylococcal virulence factors that stand out as prospective targets for immune-based therapeutics can be broadly classified into three main groups: surface molecules that lie at the interface of the organism with the host tissues, secreted toxins and exoenzymes, and factors capable of manipulating the host immune system. Candidate immunogens have been previously identified among these staphylococcal factors and tested for their ability to induce protection in either animal model systems, or, in some cases, human clinical trials. The complex bacterial surface of S. aureus forms the organism’s first line of defense against the host immune system. The cell wall of S. aureus is comprised of peptidoglycan, providing a rigid structure to the pathogen while serving as a scaffold for the attachment of a collection of bacterial proteins, lipids, and carbohydrates. Together, these surface structures play an essential role in allowing the pathogen to gain access to the host tissues. One vital class of staphylococcal surface proteins is anchored to the cell wall through the activity of the transpeptidase sortase A (SrtA) (54). The substrates of SrtA

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contain an LPXTG motif sorting signal at the C-terminus; cleavage of the surface protein between the T and G residues of the LPXTG motif allows for the generation of an acyl enzyme, which is resolved upon nucleophilic attack by the lipid II moiety. The modified surface protein attached to lipid II is subsequently incorporated into the growing cell wall. Among the SrtA-anchored proteins are several fibronectin-binding proteins (FnbA and FnbB), the fibrinogen-binding proteins ClfA and ClfB, collagen adhesin (Cna) and a collection of Sdr proteins containing serine-aspartate repeats. Collectively, these proteins bind extracellular matrix components and are known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). The iron-regulated surface determinants A and B (IsdA, IsdB) represent a second group of SrtA substrates. These proteins are also critical for S. aureus pathogenesis, participating in the uptake of iron, an essential bacterial nutrient, in the context of the iron-limiting host environment (55,56). Owing to their role in facilitating staphylococcal entry into the host tissues and nutrient acquisition, the substrates of SrtA have been considered attractive candidates for vaccine development. Most strains of S. aureus generate a polysaccharide capsule that surrounds the cell wall. Thirteen distinct serologic capsular subtypes exist, however, 80% of staphylococci produce one of two capsular polysaccharides (CPs), termed type 5 (CP5) and type 8 (CP8) (57). While structurally similar, these polysaccharides elicit the production of distinct antibodies that lack crossreactivity. These CPs likely serve to protect the organism from the phagocytic machinery of the host through the prevention of opsonophagocytosis mediated through the combined actions of antibody and complement (58–62). Strains that generate CP5 and CP8 are both more virulent than their corresponding acapsular mutants in animal models of disease (60–64). In addition to the CP, a second major surface polysaccharide, poly-N-acetylglucosamine (PNAG), has been demonstrated to play a role in S. aureus virulence. The biosynthetic machinery for PNAG is encoded by the ica locus, which is commonly present in clinical S. aureus isolates (65); deletion of this locus results in defects in virulence in murine models of bacteremia, renal abscess formation, and intraperitoneal infection (66). Interestingly, this locus is also present in Staphylococcus epidermidis, where it is known to facilitate intercellular adherence properties of the organism, thus contributing to the formation of biofilms. In S. aureus, this exopolysaccharide demonstrates increased expression in organisms grown in vivo, however, increased expression can be elicited in vitro in rich media (66). S. aureus utilizes a quorum-sensing mechanism to govern expression of its secreted toxins and surface proteins. During the late-log and stationary phases of growth, accessory gene regulator (Agr) locus is transcriptionally active, driven by the promoters P2 and P3. The P2 operon encodes a secreted autoinducer peptide, AIP. Peptide binding to its cognate cell surface receptor, AgrC, activates the response regulator AgrA (67–69); binding of this regulator to the promoter regions of the P3 operon allows for the generation of the RNAIII transcript (70). RNAIII, in turn, facilitates the upregulation of expression of staphylococcal exotoxins and a collection of proteases including the V8 and other serine proteases (71). These enzymes, along with a collection of other extracellular enzymes and the staphylococcal exotoxins mediate host tissue damage that enables bacterial spread within the tissues. Central to staphylococcal pathogenesis is the remarkable ability of the organism to breach the host defense system. A

vast array of proteins participate in this response, including staphylococcal protein A (spa), a cell wall anchored SrtA substrate that binds the Fc portion of host IgG, resulting in its precipitation (72). The CHIPS and SCIN proteins are secreted modulators of phagocyte chemotaxis and the complement system, respectively (20–22,73,74). An AMP sensing system confers resistance to these endogenous anti-staphylococcal compounds (46), while the secretion of catalase leads to the inactivation of toxic reactive oxygen species within the phagosome. Along these lines, the presence of carotenoid pigment in S. aureus also confers resistance to oxidative damage (75). Together, these immunoevasion strategies contribute to the success of S. aureus as a pathogen, and offer novel targets for vaccine-based strategies. It has been recognized for decades that the identification of virulence factors, coupled with knowledge of their mechanism of action, may direct the development of a successful vaccine against S. aureus. This strategy defines a traditional approach to vaccine development, one that has proven successful in the generation of most currently available vaccines targeting pathogens such as Bordetella pertussis, Corynebacterium diphtheriae, and Haemophilus influenzae. The availability of whole bacterial genome sequences in recent years makes possible an exciting new mechanism by which to identify new vaccine candidates. The bioinformatic analysis of multiple sequenced strains of the same species facilitates both, an appreciation of those bacterial proteins that are conserved between multiple strains and those potential vaccine candidates that are nonuniversal, leading to the identification of novel antigenic targets (76). Coupling of bioinformatics with functional genomics technologies such as DNA microarray and proteomic analysis, have proven to be a powerful tool in the identification of candidate vaccine antigens (77). To date, this ‘‘reverse vaccinology’’ approach has been successfully applied to Neisseria meningitidis and Group B streptococcus (78–80), and holds promise for a collection of other pathogens for which conventional approaches have failed to result in the development of a vaccine (80,81). At present, the complete genome sequence of 14 strains of S. aureus has been reported. The first report of a reverse vaccinology–type approach examining S. aureus was compiled by Etz et al. in 2002. This group utilized E. coli surface display technology to identify antigenic targets of human immune sera (51). Samples from S. aureus–infected patients were obtained during the acute period of illness and compared with normal sera from healthy adults for their ability to bind to expressed antigens from a S. aureus genomic library. This approach resulted in the identification of 60 staphylococcal proteins that elicited a humoral immune response in infected individuals. Among these, the majority of candidates were surfaceassociated or secreted, including staphylococcal protein A, fibronectin-binding proteins A and B, a putative exotoxin, and the extracellular enzymes lipase and coagulase. While this study did not directly assess the ability of these antigenic targets to provide immunologic protection in a vaccine-based strategy, these observations highlight the complexity of the antigenic milieu that S. aureus presents to the human host, and suggests that a collection of staphylococcal proteins may likely be of relevance for vaccine design. Further, this study highlights the power of genome-based approaches to permit the identification of antigenic targets that may not otherwise be identified through conventional strategies. While much has indeed been learned about the molecular pathogenesis of S. aureus infection, the targeting of staphylococcal

Chapter 67: Vaccines for S. aureus Infections

virulence factors has not yet led to the availability of a vaccine suitable for the human population. Nevertheless, a number of current efforts hold promise for success in the future of S. aureus vaccination.

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conferred protection against S. aureus pneumonia in laboratory animals (95). This protection was also evident upon challenge with the virulent CA-MRSA strains LAC and MW2.

Polysaccharide Vaccines VACCINE STRATEGIES TARGETING STAPHYLOCOCCUS AUREUS Protein Subunit Vaccines Early investigation into the role of staphylococcal proteins as immunogens was performed in the mid-1900s. Initial attempts involved the production of phage lysates from several S. aureus strains. These were evaluated for efficacy in the treatment of human skin infection, demonstrating an 80% recovery rate among the population tested (82). Additional protein-based vaccination strategies include further trials of protein preparations derived from either phage or enzyme-induced lysis of staphylococcal strains (83–85). More recently, whole-killed staphylococci were combined with S. aureus toxoids and examined in patients receiving renal replacement therapy via peritoneal dialysis (86). This multicenter, placebo-controlled trial demonstrated an increase in anti-staphylococcal antibodies in the peritoneal dialysis fluid, however, it was unable to demonstrate vaccine efficacy in the protection against peritonitis. Heralding from the observation that SrtA mutants of S. aureus display a virulence defect in animal models of infection (87), a number of groups have examined these proteins as vaccine candidates. Vaccines composed of the individual surface proteins ClfA (88), ClfB (89), IsdB (90), Cna (91), and FnBP (91) have all been demonstrated to confer protection against S. aureus challenge. Stranger-Jones et al. used a bioinformatics approach to guide the selection of a group of four SrtAanchored surface proteins (IsdA, IsdB, SdrD, and SdrE) that were each conserved in eight S. aureus genomes (92). Importantly, these surface proteins elicited a host antibody response upon vaccination as independent antigens. Vaccination of mice with each of these antigens in isolation afforded a modest degree of protection from renal abscess formation and mortality following S. aureus infection (92). A robust protective response was observed, however, upon the assessment of a vaccine containing a combination of all four surface protein antigens (92). Most importantly, this vaccine was able to confer protection against an array of S. aureus clinical isolates, among which are included the LAC/USA300 and MW2/USA400 strains, two extremely virulent CA-MRSA strains that account for a significant proportion of current staphylococcal infections in healthy hosts. Mechanistically, the combined surface protein vaccine yields high-titer antibody responses in the murine host; these antibodies are capable of facilitating neutrophil-mediated phagocytosis of the pathogen. The most recent approach to the generation of proteinbased anti-staphylococcal therapies grew out of the development of a murine model of S. aureus pneumonia that facilitated the identification of S. aureus a-toxin as a critical virulence factor in pathogenesis (93). This pore-forming cytotoxin exhibits some degree of specificity for erythrocytes and epithelial cells, including the alveolar epithelium that permits gas exchange in the distal lung. S. aureus mutant strains devoid of Hla expression were avirulent in the murine model of disease, and similarly, were unable to induce lytic damage to cultured alveolar epithelial cells (93,94). Active immunization with a modified, nontoxic recombinant form of the Hla protein containing a leucine for histidine substitution at residue 35 (H35L)

A single active vaccination protocol targeting S. aureus has been evaluated in phase 3 clinical trials. This vaccine, StaphVax (Nabi Biopharmaceuticals, Boca Raton, Florida, U.S.), draws on the successful immunologic approach of coupling a polysaccharide to a proteinaceous carrier. Specifically, StaphVax consists of types 5 and 8 staphylococcal CPs joined covalently to a recombinant form of Pseudomonas aeruginosa exotoxin A (rEPA) (96). These conjugates were both immunogenic in mice, generating antibodies that induced opsonophagocytic killing of S. aureus by human neutrophils (97). Immunization of healthy volunteers with StaphVax documented a greater than fourfold increase in the CP-specific antibody titers within the immunized population. The serum concentration of these antibodies reached a peak six weeks after immunization, with some decrement in specific antibody titer by six months following immunization (98). The subsequent assessment of this vaccine in phase 2 trials was performed as a multicenter project, investigating the safety and tolerability of the vaccine in endstage renal disease (ESRD) patients (99). The serum antibody responses to CPs were diminished in these patients, owing to a more rapid decrease in antibody concentration in the serum. The proven safety of this vaccine in the phase 1 and phase 2 trials prompted its examination in a multicenter, randomized double-blind trial. ESRD patients on hemodialysis were enrolled and immunized with StaphVax via a single intramuscular injection (100). The primary end point of the study was protection from invasive S. aureus disease in the one-year period immediately following vaccination. Of the 892 immunized individuals, 27 developed S. aureus bacteremia in comparison to 37 of 906 controls, a difference that failed to reach statistical significance. Among vaccinated study subjects, more than 80% developed anti-CP antibodies. Interestingly, an assessment of efficacy at the 40-week postimmunization time point did reveal a significant reduction in S. aureus bacteremia in the vaccine group relative to the control population. While the study authors observed a correlation between the decline in antibody titers and loss of protection beyond the 40-week time interval, the presence of high-level titers was not necessarily protective in any given individual. StaphVax is currently being reformulated to include not only the types 5 and 8 CP antigens but also CP336 and the two exotoxins, PVL and a-hemolysin. Studies of the role of PNAG in the pathogenesis of S. aureus infection have suggested that this exopolysaccharide may also be a suitable vaccine candidate. Indeed, mice vaccinated with PNAG developed high titers of antibody targeting this antigen, and were protected from S. aureus infection in a renal abscess model of disease (65). Together, vaccine studies on the CPs and PNAG illustrate the potentially important role these surface polymers may have in the induction of immunity to S. aureus.

Live Vaccination The origins of live vaccination against S. aureus date back to the early 1940s. Mangiaracine and Goodale of the Massachusetts Eye and Ear Infirmary described their preparation of ‘‘young (three- to four-hour)’’ cultures of S. aureus that were delivered via intramuscular injection according to a fixed five-week protocol to patients suffering from chronic ocular staphylococcal

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infection (101). Of the 78 patients treated, follow-up was obtained on 71 patients; of these, 51 showed improvement. In addition to this trial in humans, several studies have evaluated the efficacy of vaccination with live attenuated strains in animal models of disease. Live vaccination strategies in rabbits (102), goats (103), and sheep (104,105) have all demonstrated protection from staphylococcal disease. Using a murine mastitis model, immunization of pregnant or early postgestational mice with a replication-defective S. aureus mutant strain led to a reduction in bacterial recovery from the mammary gland following challenge (106). Further, the investigators observed a significant increase in the S. aureus–specific IgG and IgA titers in immunized animals (106). While controlled investigation of live vaccination strategies in humans have not been carried out in the modern era, the data obtained through these investigations suggest that the identification of an idealized, attenuated S. aureus strain may be of potential value for the future of antistaphylococcal vaccine development. In this regard, recent studies of the secretion system Ess (ESAT-6 secretion system) in S. aureus have revealed a novel mechanism by which live S. aureus induce protection in a murine model of renal abscess formation. This pathway bears homology to the ESAT-6 proteins in Mycobacterium tuberculosis. In mycobacterium, this pathway is strictly required for virulence, as strains lacking the secretion substrates ESAT-6 (esxA) and CFP-10 (esxB) are severely compromised in its ability to cause tuberculosis (107–110). In S. aureus, the Ess system facilitates the secretion of EsxA and EsxB. S. aureus strains lacking either of these proteins demonstrate a virulence defect in a murine model system, highlighting their role in pathogenesis (111). S. aureus mutants deficient in the expression of EsxB are cleared from the kidney within several days following infection of experimental animals. In spite of the more rapid clearance of this mutant, animals that receive the esxB mutant strain as a live bacterial vaccine are more resistant to S. aureus infection upon challenge with a fully virulent wild-type strain than animals that were previously infected with wild-type staphylococci (112). The molecular details of this immunoprotection require further investigation, however the study highlights the potential promise of live vaccination strategies to facilitate protection against S. aureus infection.

Passive Immunotherapies Multiple research studies have investigated the ability of purified antibodies targeting staphylococcal virulence factors to protect against disease. This approach is attractive, as it allows for the delivery of therapy when disease has already ensued, a point at which the efficacy of active vaccination is limited. Advances in monoclonal antibody technology have made this approach feasible, however, to date, no successful passive immunotherapeutics are available to combat S. aureus infection. Five anti-staphylococcal antibody therapies have been examined in clinical trials. Aurograb (Novartis AG, Basel, Switzerland) is a recombinant antibody fragment that targets an ATP-binding cassette (ABC) transporter involved in cell wall synthesis. This therapy is currently being investigated in a double-blind placebo-controlled trial in which treatment with Aurograb plus vancomycin is being compared with treatment with vancomycin alone for the management of deep-seated staphylococcal infections (113). AltaStaph (Nabi Biopharmaceuticals) hailed from observations that active vaccination with StaphVax produced a protective antibody response

against the CPs types 5 and 8 (97). These high-titer polyclonal antibodies were evaluated in a group of low birth weight infants. While demonstrating appropriate safety and pharmacokinetic profiles, the study was not powered to reveal efficacy (114). A second high-titer human polyclonal antibody that targets MSCRAMMs (bacterial proteins that bind to host extracellular matrix proteins), Veronate (INH-A21, Inhibitex), was also evaluated in low birth weight infants as part of a phase 3 clinical trial. Two randomized, placebo-controlled studies did not reveal significant effects on the primary end point of reduction in the frequency of S. aureus infection (115,116). Aurexis (Tefibazumab, Inhibitex, Georgia, U.S.) targets S. aureus clumping factor A. This monoclonal antibody was recently subjected to phase 2 trials, in which 52 patients with S. aureus bacteremia were evaluated, demonstrating a favorable safety and tolerance profile (117). Finally, a monoclonal antibody directed against lipoteichoic acid [BSYX-A110, Pagibaximab, Biosynexus, and GlaxoSmithKline (Rixensart, Belgium)] has been evaluated in phase 2 clinical trials (118). This immunotherapy was well tolerated by the population of low birth weight infants in which it was investigated. No infants in the treatment group suffered from S. aureus sepsis, compared with 13% in the placebo group. At present, additional studies are planned to evaluate the efficacy of this therapy. While not assessed in clinical trials, a number of additional passive immunization strategies have also been evaluated for their ability to confer protection against staphylococcal disease. Passive immunization of mice with rabbit antisera raised against a nontoxic a-hemolysin mutant was able to protect the animals from lethality induced by intraperitoneal injection of the purified toxin as well as live S. aureus infection (119). Similarly, passive immunization with anti-PNAG polyclonal rabbit antisera demonstrated protective efficacy in a murine model of S. aureus bacteremia and renal infection (65,120). Extension of these studies led to the development of a fully human monoclonal antibody (MAb F598) targeting PNAG; passive immunization of mice with this monoclonal conferred protection against lethal challenge with S. aureus (121). The recent observation that S. aureus a–hemolysin is essential for the pathogenesis of pneumonia in a murine model system (93) led to the investigation of the protective function of anti-Hla antibodies. Similar to results observed when animals received an active vaccination protocol, antagonism of Hla by passive transfer of immune sera conferred significant reductions in animal mortality from S. aureus pneumonia (95). Further, this protection was associated with improvements in the pathologic features of disease and a reduced bacterial recovery from the lungs of infected animals (Table 1) (95). Together, these efforts at the identification of passive immunotherapy for S. aureus infection hold promise that the field may ultimately arrive at a successful strategy. It appears clear from the studies to date, however, that the staphylococcal target, patient population, and clinical indication for such therapies will require careful definition. Such strategies are not likely to be universal in nature, yet may be of remarkable benefit to select populations.

FUTURE DIRECTIONS The challenge of developing a S. aureus vaccine with effectiveness in the human population remains unmet, in spite of the ongoing efforts of numerous research teams. This is likely due

Chapter 67: Vaccines for S. aureus Infections

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Table 1 Summary of Staphylococcus aureus Vaccine Trials Vaccine type Protein subunit

Polysaccharide

Live

Passive

Immunogen (or antibody target) S. aureus phage lysate (phage groups I/III) S. aureus phage lysate (group I) Enzyme-treated S. aureus Whole-killed S. aureus þ toxoid ClfA ClfB IsdB Cna-FnbP IsdA, IsdB, SdrD, SdrE a-hemolysin CP5, CP8 þ Pseudomonas aeruginosa exotoxin A PNAG

Species/model system

End point

End point met

Reference

Human/skin infection

Clinical recovery

Yes

82

Human/skin infection

Clinical recovery

No

85

Rabbit/skin infection

Reduction in formation of skin lesions Reduction in peritonitis

Yes

83,84

No

86

Reduction in arthritis Reduced nasal colonization Mortality reduction Mortality reduction Mortality reduction Mortality reduction Bacteremia reduction at 54 wk post immunization Reduction in renal CFU recovery Clinical improvement

Yes Yes

88 89

Yes Yes Yes Yes Noa

90 91 92 95 100

Yes

65

Yes

101

Reduction in mammary CFU recovery Reduction in renal CFU recovery Clinical and pathologic improvement Reduction in frequency of infection Reduction in frequency of infection Safety/tolerance study Reduction in S. aureus sepsis Mortality reduction

Yes

106

Yes

112

In trial

113

No

114

No

115,116

Yes Yes

117 118

Yes

119

Mortality reduction

Yes

95

Reduction in renal CFU recovery Mortality reduction

Yes

65,120

Yes

121

Human/peritoneal dialysis patients Mouse/septic arthritis Mouse/nasal colonization Mouse/sepsis Mouse/sepsis Mouse/renal abscess Mouse/pneumonia Human/hemodialysis patients Mouse/renal abscess

‘‘Young’’ culture of live S. aureus Replication-defective live S. aureus S. aureus esxB mutant

Human/chronic eye infection Mouse/mastitis

ABC transporter (Aurograb) CP5/CP8 (AltaStaph)

Human/deep-seated infection Human/low birth weight infants Human/low birth weight infants Human/bacteremia Human/low birth weight infants (i) Mouse—peritoneal infection (ii) Mouse—pneumonia

MSCRAMMs (Veronate) ClfA (Aurexis) Lipoteichoic acid a-hemolysin

PNAG

Mouse/renal abscess

(i) Mouse—renal abscess (ii) Mouse—sepsis

a

In this study, a reduction in the incidence of bacteremia was evident in immunized individuals at the 40-week time point. Abbreviations: Isd, iron-regulated surface determinants; Cna, collagen adhesin; Fnb, fibronectin-binding protein; CP, capsular polysaccharide; PNAG, poly-Nacetylglucosamine; ABC, ATP-binding cassette; MSCRAMMs, microbial surface components recognizing adhesive matrix molecules.

to the formidable complexity of the organism, its genetic plasticity that facilitates the acquisition and transfer of both virulence determinants and antimicrobial resistance, and the lack of understanding, at present, of the features of the host immune response that best contribute to eradication of the pathogen. As the vaccine community searches for an effective antistaphylococcal vaccine, it is imperative that the evaluation of candidate vaccines takes into account the dynamic epidemiology of S. aureus infection. To date, much of the thrust of S. aureus vaccinology has centered on the identification of patient populations that are at significant risk for the development of disease. It has been the prevailing view that a well-designed vaccine, when delivered to a high-risk population should mitigate a substantial burden of disease. As such, there has been

limited attention given to the development of a ‘‘universal’’ vaccine. The dramatic changes in the epidemiology of S. aureus infection in the past decade have amply demonstrated that we are all at risk for the development of invasive staphylococcal disease. These epidemiologic changes have thereby ushered in a new era of S. aureus vaccine development, requiring a thoughtful delineation of the goals for S. aureus vaccination. Along with a careful appraisal of the type of vaccine that is sought after, it is imperative that we obtain detailed knowledge of the mechanism by which efficacious vaccines augment or direct the host response. It is clear that humans do not reliably develop long-term protective immunity to S. aureus. Therefore, defining the constraints of the natural host response to S. aureus infection, in concert with an appreciation of the idealized response derived from successful vaccination of model animals,

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can be envisioned to provide a foundation on which to optimize the elicited response to a vaccine. Advances in our understanding of the immunostimulatory properties of novel adjuvants will likely facilitate the design of an anti-staphylococcal vaccine that optimizes the host response to the vaccine. The rapid spread of antimicrobial resistance among virulent S. aureus strains in the past decade has outstripped the development of novel drugs with anti-staphylococcal potency. At the same time, the progression toward the development of anti-staphylococcal vaccines has been slow. It is evident that the collective efforts of clinicians and researchers alike will be required to curb the spread of this virulent organism, halt the progression of antimicrobial resistance, and ultimately, arrive at a vaccine strategy that offers protection to the human population.

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68 Chlamydia trachomatis Vaccines Renata Grifantini Externautics, Siena, Italy

Guido Grandi Novartis Vaccines and Diagnostics, Research Center, Siena, Italy

INTRODUCTION Chlamydiae are obligate intracellular gram-negative bacteria with a highly specialized biphasic life cycle that alternates between two developmental forms, the elementary body (EB) and the reticulate body (RB) (1) (Fig. 1). EBs, the small form of the bacterium (300 nm), survive in the extracellular environment, display little or no metabolic activity, and represent the infectious form of the organism. Upon contact with host cells, EBs induce their own uptake, termed ‘‘inclusion,’’ into a nonacidified intracellular vacuolar compartment, where, within two hours, they are converted into metabolically active, noninfectious replicative forms called RBs. The RBs then undergo several rounds of replication by binary fission within the expanding inclusion, and after about 18 hours they begin to differentiate back into EBs. Depending on the Chlamydia serovar, at approximately 36 to 72 hours from initial infection, cell lysis occurs and EBs are released into the extracellular space where they can infect other cells and begin a new replicative cycle. Three major species belonging to the Chlamydiaceae family are of relevance in human pathology: Chlamydia psittaci, a species that usually infects birds but can cause pneumonialike disease and severe systemic illness in humans; C. pneumoniae, responsible for up to 10% of all community-acquired pneumonia and considered to be associated to coronary heart disease; and C. trachomatis. This pathogen is classified in 19 different serotypes (serovars) on the basis of the expression of distinct variants of its major outer membrane protein (MOMP). Certain serovars are associated with infection of ocular tissues, while other serovars are associated with infection of genital tissues (2,3). In particular, serovars A–C infect the conjunctival epithelium and can give rise to trachoma, the leading cause of preventable blindness worldwide (4). Serovars D–K infect ophthalmic, rectal, and genital columnar epithelial cells, causing conjunctivitis, urethritis, cervicitis, and proctitis, respectively. These serovars also infect respiratory epithelial cells and cause infant pneumonitis. Finally, serovars L1–L3 also establish infection in the urogenital tract but can additionally spread into the draining lymph nodes to cause a relatively rare systemic disease of the lymphatic system called lymphogranuloma venereum. C. trachomatis represents a global health problem. Approximately 100 million people are infected every year worldwide. Sexually active young women between 16 and 19 years and men between 20 and 24 years have the highest prevalence of

chlamydial genital infection in industrialized countries. In up to 30% of infected women, low genital tract infections ascend the endometrial epithelium and extend to the fallopian tubes, where C. trachomatis can establish persistent infection, causing pelvic inflammatory disease (PID). It has been estimated that approximately 15% of all C. trachomatis–associated PID leads to infertility, while up to 9% of cases results in ectopic pregnancies (5). In developing countries, C. trachomatis is responsible for a large number of eye infections. The WHO has estimated that approximately 146 million individuals have C. trachomatis– associated trachoma, and six million people are affected by trachoma-induced blindness. Finally, recent studies indicate that infection with C. trachomatis facilitates the transmission of HIV and might be a cofactor of human papilloma virus (HPV)induced cervical neoplasia (6,7). Although effective antibiotics are available, such as azithromycin or doxycycline, for a number of reasons antibiotic therapy appears to be inadequate in halting the spread of infection and preventing Chlamydia-associated long-term sequelae. First, urogenital tract infections are often asymptomatic and therefore not properly treated in due time. Second, the emergence of multiple antibiotic resistance has been reported in an increasing number of Chlamydia isolates (8). Third, there are indications from in vitro studies that antibiotic treatment could facilitate the formation of aberrant Chlamydia forms that remain dormant in infected individuals and eventually turn into RB under favorable environmental conditions (9,10). In view of the high prevalence of chlamydial infection, many industrialized countries are undertaking widespread screening programs for the early diagnosis of C. trachomatis infections and to allow effective antimicrobial treatment of both infected individuals and their sexual partner(s) (11). Although these programs have the potential to prevent the spread of infection, they are expensive and, to be efficacious, they should include the entire population at risk for a prolonged period of time. At present, epidemiological data indicate that in those countries where screening campaigns are in progress, the incidence of reported Chlamydia-associated diseases is only marginally decreasing but the rate of Chlamydia reinfection is rising. A plausible explanation for this increase is that screening campaigns are not fully satisfactory and early administration of antibiotics prevents the establishment of natural immunity with the consequence that antibiotic-treated patients are easily reinfected once reexposed to Chlamydia (11,12).

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Figure 1 Schematic representation of the evolution of Chlamydia trachomatis infection in the presence (right panel) or absence (left panel) of adaptive immune responses. In naı¨ve individuals (left panel), chlamydial EBs infect epithelial cells of the lower genital tract where they multiply within inclusions and rapidly spread. Eventually, EBs reach the female upper genital tract and, under particular conditions, cause chronic infections (for the sake of simplicity, innate immune responses are not reported in the figure). An ideal vaccine should elicit an adaptive immune response (right panel), which, from the one hand, should involve antibodies capable of neutralizing EBs infection. Antibodies should also synergize with cell-mediated immunity by enhancing antigen presentation to T cells via an Fc-receptor-mediated uptake of antigen-antibody complexes (not shown in the figure). On the other hand, antigen-presenting cells should activate chlamydialspecific T cells, which, in turn, should eliminate Chlamydia-infected cells through interferon-g-mediated mechanisms. Abbreviations: EB, elementary body; RB, reticulate body; IFN-g, interferon g.

Given the above, vaccination appears to be the most efficacious and long-lasting strategy to fight C. trachomatis infections and C. trachomatis–associated diseases. Unfortunately, the development of an effective vaccine is proving to be challenging. Indeed, apart from vaccination attempts reported

in the 1970s in which whole inactivated EBs were used (13), no vaccine candidates so far have reached the point of entering clinical trials. However, it is expected that our better understanding of Chlamydia biology and immunology, together with the availability of powerful genomic and proteomic

Chapter 68: Chlamydia trachomatis Vaccines

technologies, will accelerate the pace of C. trachomatis vaccine research in the near future. This chapter provides an overview of the major efforts and achievements in the C. trachomatis vaccine field, highlighted in the context of our current knowledge of chlamydial immunology.

IMMUNE RESPONSES TO C. TRACHOMATIS INFECTION As a general rule in vaccinology, the success in developing a vaccine against a particular pathogen is directly correlated to the level of natural immunity the host acquires after primary infection. Although recurrent Chlamydia infections are well documented, there is a sufficiently large body of evidence supporting the notion that C. trachomatis does elicit natural immunity in humans. Such evidence is mostly derived from epidemiological studies and from human challenge experiments and vaccine trials. As far as epidemiological evidence is concerned, as already mentioned, the incidence of sexually transmitted, Chlamydia-associated diseases peaks in human population ranging from 15 to 25 years and rapidly drops afterward (4). Considering that people older than 25 years are sexually active, these epidemiological data are generally interpreted as demonstration of the existence of natural immunity. Furthermore, it has been shown that among attendees of sexually transmitted disease (STD) clinics, the risk of C. trachomatis infection was significantly reduced among those with a history of STD or previously documented chlamydial infection (14). Finally, the risk of reinfection is higher in people living in countries adopting Chlamydia screening programs whose infection has been promptly diagnosed and treated with antibiotic therapy (11). Evidence derived from human challenge experiments and results of vaccine trials reported by Jawetz et al. (15) convincingly demonstrate resistance to ocular infection in human subjects. Nine volunteers with previous C. trachomatis infection were challenged with a chlamydial infective dose and 53% of them were fully protected. Interestingly, the protected volunteers were those who were challenged with the homologous strain, while volunteers challenged with a heterologous strain were all infected. In the same study, volunteers without known previous exposure to Chlamydia were also challenged and 93% of them became infected. Vaccine trials also demonstrated that immunization with C. trachomatis elicits protective immunity. Grayston and Wang (13) reported a vaccination campaign of 332 children, living in areas at risk of active trachoma, who received intramuscularly either inactivated chlamydial cells or placebo. During the first year of followup, the vaccine group showed up to 73% reduction in trachoma incidence. A second important factor that usually facilitates vaccine development is the knowledge of the immune correlates of protection. Most of our current knowledge of the correlates of protection in Chlamydia immunity derives from the numerous studies performed over the last few decades in the mouse model of vaginal infection using the Chlamydia muridarum strain. This model, that makes use of a mouse-adapted C. trachomatis strain sharing an almost identical genetic makeup with the human strain, closely mimics acute infection of the genital tract in women (16,17). Thanks to this model, as well as to human studies, it has been unequivocally demonstrated that Chlamydia immunity correlates with a strong T helper cell type 1 (Th1) response and a complementary antibody

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response that fosters a rapid and robust memory T-cell-mediated immunity. The crucial role of T cells has been demonstrated in several studies. For instance, nude mice cannot control Chlamydia infection but when these mice are given Chlamydiaspecific T-cell lines, they successfully control infection (18–21). In particular, in the C. muridarum infection model, protection appears to be mediated by interferon-g (IFN-g)-producing CD4þ T cells, as mice deficient in major histocompatibility complex (MHC) class II molecules, CD4, interleukin-12 (IL12), IFN-g, or the IFN-g receptor and mice depleted of C. muridarum–specific CD4þ T cells all have a marked inability to control infection (22–29). Furthermore, adoptive transfer of CD4þ Th1-cell clones, but not CD4þ Th2-cell clones, protects nude mice against infection (30). The role of CD8þ T cells has not been completely clarified. Antigen presentation to MHC class I does not appear to be required for immunity to Chlamydia infection, since mice deficient in relevant molecules of CD8þ T cells, such as b2-microglobulin, CD9, and perforin, are still able to resolve Chlamydia infection (23,24,31). However, adoptive transfer of IFN-gproducing Chlamydia-specific CD8þ T cells into naı¨ve mice confers protection against C. trachomatis challenge and CD8þ T-cell-dependent cytolysis of Chlamydia-infected cells has been observed ex vivo, indicating that CD8þ T cells could eliminate Chlamydia-infected cells (21,32,33). The mechanism by which both CD4þ and CD8þ T cells mediate protection against Chlamydia infection appears to be largely mediated by IFN-g. This inflammatory chemokine promotes Chlamydia clearance through both indirect and direct mechanisms. IFN-g can activate the phagocytic activity of macrophages, promoting EB engulfment and destruction (34). IFN-g can also inhibit Chlamydia growth within infected cells by inducing nitric oxide synthase (35,36) and can limit intracellular iron storage through downregulation of the transferring receptor (37,38). Furthermore, IFN-g is known to induce the expression of indoleamine 2,3-dioxygenase, an enzyme that leads to the degradation of tryptophan, and the lack of this essential amino acid results in Chlamydia death through tryptophan starvation (39). It has been recently shown that the genital C. trachomatis serovars, but not the ocular serovars, can synthesize tryptophan from indole. Although Chlamydia cannot produce indole, it can utilize the indole eventually released by the microbial flora living in the genital tract. The capacity of C. trachomatis to synthesize tryptophan when it is limited in the host cells has been reputed as an evasion mechanism that counteracts the tryptophan degradation induced by IFN-g (40,41). The importance of B cells and antibodies in C. trachomatis immunity originally emerged from early epidemiological studies showing that the presence of secretory immunoglobulin A (IgA) in the genital mucosa inversely correlated with the C. trachomatis load recovered from the cervix of infected women (42). However, studies on the protective role of B cells in the mouse model showed that B-cell-deficient mice control primary Chlamydia genital infection as efficiently as wild-type mice, indicating that B cells do not play a critical role during primary infection (43). More recent observations in animal models demonstrated that B cells have, however, a role in the resolution of secondary Chlamydia infection. B-cell-deficient mice depleted of CD4þ T cells are completely unable to control secondary infection, whereas wild-type, CD4þ T-cells-depleted mice are still capable of clearing secondary infection (24). This B-cell-mediated protection is due to

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antibody production since B-cell deficient or CD4 þ T-cell-depleted mice receiving anti-Chlamydia immune serum or Chlamydia-specific monoclonal antibodies recover the ability to control secondary infection (44). Antibodies could contribute to Chlamydia immunity by both neutralization and opsonization mechanisms. Monoclonal antibodies against MOMP neutralize infection in vitro (45,46) and provide a partial level of protection against infection when passively administered to mice (47,48). In addition, there is evidence that antibodies synergize with cell-mediated immunity enhancing antigen presentation to T cells via an Fc-receptormediated uptake of antigen-antibody complexes (49). In conclusion, data in mouse models as well as recent studies in nonhuman primates and in C. trachomatis infected patients show that CD4þ-Th1 cells are key for the resolution of Chlamydia infection. CD8þ T cells seem to have a supporting role and antibodies appear to be relevant in secondary infection and in enhancing chlamydial antigen presentation via an Fc-receptor-mediated process.

C. TRACHOMATIS VACCINE DEVELOPMENT Initial human vaccine trials using heat-inactivated preparations of whole EBs represent an important proof-of-concept of antiChlamydia vaccines. However, vaccination seemed to exacerbate the disease during reinfection episodes in few individuals (13). For this reason, vaccine discovery efforts have been focusing on the identification of chlamydial protective antigens which, in combination with appropriate adjuvants and/or delivery systems, could induce a Th1-polarized immune response and, in particular, IFN-g-secreting CD4þ T lymphocytes. To select protective antigens, the C. muridarum mouse model with three different routes of challenge has been largely utilized. According to the two most popular routes, mice (BALB/c or C57BL/6) are given 103 to 104 EBs either intranasally or intravaginally, and infectious forming unit (IFU) numbers are counted in lung homogenates and vaginal swabs, respectively, over three to four weeks post challenge. Typically, after challenge C. muridarum starts multiplying and reaches approximately 105 to 107 IFUs within 10 days. At day 11–14, IFU counts decline and infection is usually cleared within 21 to 28 days post infection. A third challenge model involves intraovarian bursa infection, followed by analysis of chlamydial shedding in the lower genital tract and measurement of the level of infertility provoked by the challenge. Because of its complexity, this model is used only in few laboratories and is not amenable for high throughput antigen selection. Whichever challenge model is used, primary infection elicits natural immunity and mice are protected from a second challenge: IFUs do not increase after challenge, bacteria disappear within 10 days, and challenged mice are as fertile as controls. Because of its efficacy in protecting against secondary infection, primary infection is generally used as the gold standard positive control in vaccine discovery. The effectiveness of a vaccine candidate is measured by comparing its protective activity with the protection achieved by primary infection. By using the C. muridarum model, a number of vaccine formulations have been reported to confer partial protection against chlamydial challenge. On the basis of the protein antigen used, these vaccine formulations can be grouped in three major categories: MOMP-based vaccines, non-MOMPbased vaccines, and vaccines constituted by combinations of chlamydial antigens (Table 1).

MOMP Vaccines MOMP accounts for 60% of the total mass of the Chlamydia outer membrane. Different MOMP-topological models have been proposed, using algorithms predictive of secondary structures. According to these models, MOMP is an intrinsic membrane protein potentially bearing 16 to 18 transmembranespanning segments forming a barrel-like structure typically found in bacterial porins (50–52). The protein is highly immunogenic in both humans and animal models, being the target of both humoral and cellular immune responses. It elicits both antibodies capable of neutralizing Chlamydia infection in vitro (45,47) and T cells (53–57). For this reason, MOMP has been the first antigen to be tested as subunit vaccine. Several studies describe the use of MOMP as vaccine candidate. In general, these studies consistently show that MOMP is a protective antigen but the level of protection varies depending on the method used for its production and the adjuvant/formulation used in immunization. In particular, native MOMP purified from C. muridarum EBs conferred protection against intrabursal challenge when formulated with Freund’s adjuvant (58) and, even more efficiently, with Montanide ISA 720 (an oil-based adjuvant used in human studies for malaria vaccine) coupled to CpG (59–61). Lower protection levels were obtained when MF59, or the heat-labile enterotoxin mutants LTK63 and LTR72 were used as adjuvants (62). Attempts to use recombinant MOMP instead of EB-purified MOMP have been in general less successful, in that only weak or partial protection was reported. This has been attributed to the fact that in Escherichia coli MOMP is expressed as inclusion bodies and no efficient methods for MOMP refolding have been described so far. Considering that unfolded proteins should be as good as their native form in inducing CD4þ T-cell-mediated responses, these data would support the notion that in addition to cell-mediated immunity, anti-MOMP antibodies also play a role in protection. In line with this conclusion are the recent data of Hansen and coworkers (63) who reported that recombinant MOMP formulated with CAF01 (cationic liposomes that polarize a strong Th1 response) (64), effectively controls chlamydial replication in vaccinated mice but the same mice still show signs of pathology six weeks after challenge. The authors conclude that specific anti-MOMP antibodies may promote increased uptake of antibody-coated chlamydial EBs in Fc-receptor-positive antigen-presenting cells, leading to accelerated triggering of T-cell immunity and rapid clearance of Chlamydia. Finally, it is worth mentioning that DNA immunization, which also elicits a strong Th1 response, provided protection in the lung model of Chlamydia infection (65) when boosted with protein MOMP formulated with the cationic immunostimulatory complexes ISCOMs. In conclusion, MOMP is a promising protective antigen. However, since proper folding appears to be required to elicit robust and consistent protection, scalable methods of MOMP production in its native conformation need to be developed. At present, this seems to be a severe obstacle. Another possible problem in the use of MOMP as vaccine is associated with its sequence variability. As pointed out earlier, MOMP exists in 19 major alleles, which are serologically different. Although, on the basis of the sequence homology among alleles, it has been postulated that a vaccine constituted by four MOMP alleles should be sufficient to provide broad cross-protection (59), the development of a vaccine exclusively constituted by MOMP remains a challenge.

BALB/c BALB/c CH37HeN BALB/c

C3H/HeN C57BL/6

C57BL/6 BALB/c

None ISCOMs OspA Cholera toxin þ CpG CAF01 (Cationic Liposomes) rVCG IL-12 CpG LTR192G rVCG None None IL-12 Vaccinia virus rVCG rVCG IL-12

MOMP (DNA) MOMP (DNAProtein) MOMP

MOMP

MOMP CPAF

CPAF

Polymorphic protein G PorB

TC0512 (DNA) Pgp3 (DNA)

IncA Cysteine-rich protein A MOMPþ Omp2

MOMPþ PorB

MOMPþ CPAFþ IncA

C57BL/6

BALB/c C57BL/6

BALB/c C3H/HeN

BALB/c

C57BL/6 BALB/c

C57BL/6

BALB/c

Montanide þ CpG

Native MOMP

MOMP

BALB/c

Freund’s

Native MOMP

Intramuscular

Intramuscular

Intramuscular

Intramuscular Intraperitoneal

Epidermal Intradermal

Intramuscular

Intranasal

Intranasal

Intramuscular Intranasal

Subcutaneous

Intramuscular and subcutaneous Trancutaneous

Intramuscular Intramuscular

Intramuscular

Intramuscular

Immunization route Upper genital tract Upper genital tract Intranasal Intranasal Vaginal

C. muridarum

C. muridarum C. muridarum C. muridarum Vaginal Vaginal

Vaginal Vaginal Vaginal Upper genital tract Vaginal Vaginal Vaginal Vaginal Venous Vaginal Vaginal Vaginal

C. muridarum C. muridarum C. trachomatis C. muridarum C. muridarum C. trachomatis C. trachomatis C. muridarum C. trachomatis C. muridarum C. trachomatis C. trachomatis C. trachomatis C. muridarum

C. muridarum

Challenge route

Challenge species

Accelerated clearance of infection and 30% increase of fertility rate Reduced Chlamydia shedding in vagina Reduced Chlamydia shedding (56% of protected mice) Reduced Chlamydia shedding in vagina *1.4 log reduction of Chlamydia shedding in the spleen Reduced Chlamydia shedding (80% of protected mice) Accelerated clearance of infection and 50% increase of fertility rate Accelerated clearance of genital infection and reduced oviduct pathology

Accelerated clearance of infection and protection against genital tissue pathology Accelerated clearance of infection Accelerated clearance of infection and reduced oviduct pathology Reduced Chlamydia shedding and oviduct pathology 70% increase of fertility rate

*50% IFU reduction in vagina

*50% IFU reduction in vagina

Resolution of Chlamydia infection. Protection similar to that conferred by Chlamydia primary infection *103 IFU reduction in lungs *106 IFU reduction in lungs

*70% IFU reduction in vagina

Protection

78

71

70

78 74

72 73

71

68

91

71 69

63

90

89

65 65

59

58

Reference

Abbreviations: MOMP, major outer membrane protein; CPAF, chlamydial protease-like activity factor; CpG oligonucleotide; OspA, outer surface protein A of Borrelia burgdorferi; rVCG, recombinant Vibrio cholerae ghosts; IL, interleukin.

Multisubunit vaccines

Non-MOMP vaccines

MOMP vaccines

Mouse strain

Adjuvant/delivery system

Vaccine

Table 1 Chlamydial Protective Antigens in Active Immunization

Chapter 68: Chlamydia trachomatis Vaccines 741

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Non-MOMP Vaccines The intrinsic limits of MOMP as vaccine have fostered the search for other protective antigens. However, although the list of chlamydial antigens shown to be immunogenic both in humans and in animal models is rapidly growing (11,66–68), those demonstrated to be protective in active immunization are relatively few. They include three proteins that are found on the chlamydial surface [the outer membrane protein TC0512, porinB (PorB), and the polymorphic protein G (PmpG)], one protein secreted into the cytoplasm of Chlamydia-infected host cells (CPAF), two proteins associated with the inclusion membrane [cysteine-rich protein A (CrpA) and inclusion protein A (IncA)] and the periplasmic plasmid-encoded protein 3 (Pgp3). In particular, the recombinant chlamydial protease-like activity factor (rCPAF), when administered in combination with the Th1 cytokine IL-12, significantly reduced C. muridarum vaginal shedding and oviduct pathology in vaccinated mice (69). The same authors showed that IL-12-IncA formulation also reduced vaginal shedding, albeit to a lesser extent than IL-12-CPAF. As far as PorB is concerned, intramuscular immunization with PorB-expressing Vibrio cholerae ghosts [rVCG, dead bacteria devoid of cytoplasmic content but maintaining their native surface antigenic structure and cellular morphology (70)] resulted in accelerated resolution of vaginal infection and reduced infection-mediated infertility (71). TC0512 was identified by screening plasmid libraries of C. trachomatis genes in DNA immunization experiments. The deconvolution of a pool of plasmids conferring partial protection from intravaginal challenge led to the selection of TC0512 that showed a 73% reduction in bacterial shedding (72). DNA immunization was also used to demonstrate the partial protection conferred by Pgp3, a periplasmic protein encoded by a C. trachomatis plasmid and known to elicit a strong antibody response in both infected humans and mice (73). The PmpG, administered intranasally with the mucosal adjuvant LTR192G (74), conferred 70% protection against infertility in mice receiving a C. trachomatis intrabursal infection (68). Finally, CrpA, a protein associated to the inclusion membrane, has been described as a target of CD8þ T cells. Intraperitoneal immunization with Vaccinia virus expressing CrpA significantly reduced chlamydial load in the spleen of intravenously challenged mice compared with mice immunized with the viral vector (75). Many antigens eliciting specific B- and T-cell responses during C. trachomatis infection in both mice and humans have been described. They include the outer membrane protein 2 (Omp2), the 60 KDa heat shock protein (Hsp60), Enolase, the Yop D homologue CT579, the polymorphic protein D (pmp D), the hypothetical protein CT788, Cta1, CrpA, and the ribonucleotide reductase small chain protein NrdB (11,75,76). Although some of them were shown to elicit protective CD4þ and CD8þ T cells, as established by adoptive transfer experiments, their protective activity in active immunization remains to be demonstrated.

Multisubunit Vaccines Milestone studies with bone marrow–derived dendritic cells (DCs) have shown that the adoptive transfer of DCs pulsed with inactivated EBs but not with MOMP fully protect mice against chlamydial infection of the genital tract (77). These results seem to indicate that a large repertoire of T- and B-cell populations specific for a battery of chlamydial antigens needs to be induced to mount strong, long-lasting protection against

Chlamydia. For this reason, in the last few years, researchers have started to investigate combinations of two or more chlamydial antigens. At present, the protective activity of three combinations have been reported in the literature. Li and coworkers evaluated the combination of CPAF, MOMP, and IncA, coadministered intranasally with IL-12, and compared the protection level of the combo with the protection achieved with CPAFþIL-12 alone. Although the three-antigen combination was protective, the presence of MOMP and/or IncA did not substantially improve the efficacy of CPAF alone (78). The absence of an additive effect could be partially explained by the fact that recombinant rather than native MOMP was used in this study. In the other two vaccine combinations, MOMP was coexpressed in V. cholerae ghosts with either Omp2 or PorB (70,71). Both rVCG-MOMP-PorB and rVCG-MOMP-Omp2 combinations elicited a higher level of protection than rVCGsMOMP and rVCG-PorB. Protection correlated with stronger cellular and humoral immune responses. Particularly promising results were seen with the rVGC-MOMP-PorB vaccine that accelerated the resolution of infection and largely prevented infertility of vaccinated mice (71).

FUTURE PERSPECTIVES Will an anti-Chlamydia vaccine become available in the near future? There are reasons for both optimism and concern. The data accumulated heretofore have shown that when proper adjuvants and antigens are combined, significant protection in animal models can be achieved, even though protection levels have not yet reached the levels obtained with the gold standard positive control (primary infection). A list of protective antigens is now available and new antigens are expected to be uncovered soon through the application of genomics and proteomics. For instance, using a reverse vaccinology approach (79), Montigiani et al. (80) have recently identified 53 proteins localized on the surface of C. pneumoniae EBs. Of these, six were shown to elicit antibodies neutralizing C. pneumoniae infectivity in vitro (81) and five of them proved to be partially protective in the hamster (81) or mouse (82) models. In a second study, scanning the C. trachomatis genome for predicted T-cell epitopes, nine chlamydial antigens carrying epitopes shared by both human and mouse have been characterized. Among them, a promising antigen was identified able to prime CD4þ T cells that protected against a Chlamydia challenge in adoptive transfer experiments (76). Finally, by exploiting the availability of C. trachomatis transcriptional pattern during infection, Olsen et al. (66) selected highly transcribed genes as well as genes localized in proximity and within the plasticity zone. By analyzing the immune responses of patients against the proteins encoded by the selected genes, they have discovered novel antigens inducing strong cellular and/or humoral responses. The availability of a pool of protective antigens to be combined in a single vaccine formulation is likely to be of paramount importance for the design of effective vaccines against Chlamydia. The combination antigen strategy can achieve broad serovar cross-protection by activating an optimal repertoire of T and B cells. Another important factor that can accelerate the development of anti-Chlamydia vaccines is the availability of well-established infection models in guinea pigs, pigs, and nonhuman primates (83). These models can validate the vaccine candidates selected in the murine model and can be

Chapter 68: Chlamydia trachomatis Vaccines

exploited for toxicity studies, which might be particularly demanding because of the few disease exacerbation cases reported in early vaccine trials with inactivated EBs. Finally, epidemiological data show high Chlamydia infection rates in particular cohorts of people such as military personnel and college-aged girls (in whom the annual rate of infection can be as high as 10–15%). Populations with such high incidence rates will allow efficacy trials to be designed and carried out. These positive aspects are counteracted by two major hurdles. First, the success of an anti-Chlamydia vaccine will most likely require the use of an adjuvant/delivery system to stimulate a potent Th1 immune response. Even MOMP, one of the best candidates so far described, when formulated with different adjuvants performs quite differently, with protection varying from very high (as is the case of MOMP formulated with CAF01) to almost nonexistent (for instance, MOMP with alum). Unfortunately, the number of approved adjuvants is currently very limited and, because of the foreseen potential risks of using potent immune stimulators, Regulatory Authorities are extremely cautious in approving new adjuvants. The result is that so far alum is the only adjuvant accepted in the United States, and four adjuvants, alum, Novartis’s MF59 (84) and GSK’s AS04 and AS03 (85–87) are the only ones that have been approved in Europe. It is difficult to predict whether or not one of these adjuvants would be adequate for a Chlamydia vaccine. Should this not be the case and should one of the adjuvants currently in development be necessary (85–88), the availability of a Chlamydia vaccine might be substantially delayed. The second major hurdle is related to the fact that so far Chlamydia vaccine has not been included in the priority list of most vaccine companies. This is largely due to the fact that, despite the severity of the disease burden and the profound social, clinical, and economical impact of chlamydial infections, Chlamydia is not perceived by health authorities and policy makers as a major priority. The result is that a chlamydial vaccine is unlikely to be recommended for broad use by national health authorities and therefore its economic value solely relies on the more economically tenuous private market. In view of the epidemiological data showing that in industrialized countries chamydial infections start to occur at 12 to 14 years, when the young population becomes sexually active, an anti-Chlamydia vaccine should be targeted to adolescents. Considering the recent data indicating that chlamydial infection might be a cofactor of HPV-induced cervical neoplasia (6,7), HPV and Chlamydia vaccines would be an attractive combination of the future. The existence of a highly effective HPV vaccine universally utilized in adolescents might become a key factor to renew the interest of both health authorities and industry in chlamydial vaccine research.

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Chapter 68: Chlamydia trachomatis Vaccines

66.

67.

68. 69.

70. 71.

72.

73.

74.

75.

76.

77.

78.

with increased immunoglobulin A and TH1 cellular immune responses. Infect Immun 2000; 68:3074–3078. Olsen AW, Follmann F, Højrup P, et al. Identification of human T cell targets recognized during Chlamydia trachomatis genital infection. J Infect Dis 2007; 196:1546–1552. Follmann F, Olsen AW, Jensen KT, et al. Antigenic profiling of a Chlamydia trachomatis gene-expression library. J Infect Dis 2008; 197:897–905. Hafner LM, McNeilly C. Vaccines for Chlamydia infections of the female genital tract. Future Microbiol 2008; 3:67–77. Murthy AK, Chambers JP, Meier PA, et al. Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production. Infect Immun 2007; 75:666–676. Eko FO, He Q, Brown T, et al. A novel recombinant multisubunit vaccine against Chlamydia. J Immunol 2004; 173:3375–3382. Ifere GO, He Q, Igietseme JU, et al. Immunogenicity and protection against genital Chlamydia infection and its complications by a multisubunit candidate vaccine. J Microbiol Immunol Infect 2007; 403:188–200. McNeilly CL, Beagley KW, Moore RJ, et al. Expression library immunization confers partial protection against Chlamydia muridarum genital infection. Vaccine 2007; 25:2643–2655. Donati M, Sambri V, Comanducci M, et al. DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlamydial infection from the lower to the upper genital tract in C3H/HeN mice. Vaccine 2003; 21:1089–1093. McCluskie MJ, Weeratna RD, Clements JD, et al. Mucosal immunization of mice using CpG DNA and/or mutants of the heatlabile enterotoxin of Escherichia coli as adjuvants. Vaccine 2001; 9:3759–3768. Starnbach MN, Loomis WP, Ovendale P, et al. An inclusion membrane protein from Chlamydia trachomatis enters the MHC class I pathway and stimulates a CD8þ T cell response. J Immunol 2003; 171:4742–4749. Barker CJ, Beagley KW, Hafner LM, et al. Vaccine In silico identification and in vivo analysis of a novel T-cell antigen from Chlamydia 2008; 26:1285–1296. Su H, Messer R, Whitmire W, et al. Vaccination against chlamydial genital tract infection after immunization with dendritic cells pulsed ex vivo with nonviable Chlamydiae. J Exp Med 1998; 188:809–818. Li W, Guentzel MN, Seshu J, et al. Induction of cross-serovar protection against genital chlamydial infection by a targeted multisubunit vaccination approach. Clin Vaccine Immunol 2007; 14:1537–1544.

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79. Rappuoli R. Reverse vaccinology. Curr Opin Microbiol 2000; 3:445–450. 80. Montigiani S, Falugi F, Scarselli M, et al. Genomic approach for analysis of surface proteins in Chlamydia pneumoniae. Infect Immun 2002; 70:368–379. 81. Finco O, Bonci A, Agnusdei M, et al. Identification of new potential vaccine candidates against Chlamydia pneumoniae by multiple screenings. Vaccine 2005; 23:1178–1188. 82. Thorpe C, Edwards L, Snelgrove R, et al. Discovery of a vaccine antigen that protects mice from Chlamydia pneumoniae infection. Vaccine 2007; 25:2252–2260. 83. Vanrompay D, Lyons JM, Morre´ SA. Animal models for the study of Chlamydia trachomatis infections in the female genital infection. Drugs Today Barc 2006; 42(suppl A):55–63. 84. Frey S, Poland G, Percell S, et al. Comparison of the safety, tolerability, and immunogenicity of a MF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccine in non-elderly adults. Vaccine 2003; 21:4234–4237. 85. Baldridge JR, McGowan P, Evans JT, et al. Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents Expert Opin Biol Ther 2004; 4: 1129–1138. 86. Del Giudice G, Rappuoli R. Adjuvants and subunit vaccines. In: Kaufmann S, Steward M, eds. Topley and Wilson’s Microbiology and Microbial Infections. 10th ed. London: Arnold, 2003:926–946. 87. Pink JR, Kieny MP. 4th meeting on Novel Adjuvants Currently in/ close to Human Clinical Testing World Health Organization— organisation Mondiale de la Sante´ Fondation Me´rieux, Annecy, France, June 23–25, 2003. Vaccine 2004; 22:2097–20102. 88. Kanzler H, Barrat FJ, Hessel EM, et al. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med 2007; 13:552–559. 89. Pal S, Luke CJ, Barbour AG, et al. Immunization with the Chlamydia trachomatis major outer membrane protein, using the outer surface protein A of Borrelia burgdorferi as an adjuvant, can induce protection against a chlamydial genital challenge. Vaccine 2003; 21:1455–1465. 90. Berry LJ, Hickey DK, Skelding KA, et al. Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection. Infect Immun 2004; 72:1019–1028. 91. Cong Y, Jupelli M, Guentzel MN, et al. Intranasal immunization with chlamydial protease-like activity factor and CpG deoxynucleotides enhances protective immunity against genital Chlamydia muridarum infection. Vaccine 2007; 25:3773–3780.

69 Malaria Vaccines in Clinical Development: Introduction and Recombinant/Subunit Approaches Laura B. Martin Novartis Vaccines Institute for Global Health, Siena, Italy

Robert W. Sauerwein and Teun Bousema Department of Medical Microbiology, Radbound University Nijmegen Medical Center, Nijmegen, The Netherlands

David E. Lanar and Thomas L. Richie U.S. Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, Silver Spring, Maryland, U.S.A.

INTRODUCTION TO MALARIA VACCINES Few challenges in public health exceed the importance, or the difficulty, of developing a vaccine against malaria. The beneficial impact that would result from an intervention providing solid, long-lasting protection against this ancient affliction would be enormous (1), considering that there are 300 to 660 million clinical cases of Plasmodium falciparum each year (2,3), and that over three billion of the world’s people live at risk from the bite of a female Anopheline mosquito harboring this parasitea (6). Transmission is so intense in parts of sub-Saharan Africa that the entire population is, on average, reinfected multiple times every night (7,8). The problem is compounded by the fact that no individuals living in endemic areas acquire sterile immunity, even following years of exposure (9). The threat faced by those living in endemic areas extends to millions of travelers, who may face malaria as their single greatest health risk when visiting the tropics, if one takes into account the inapparency of the vector’s approach, the ease of transmission, and the severity of the ensuing disease (10–13). Moreover, malaria suppresses the economic development of endemic countries (14,15), as illustrated by the toll taken during major development projects such as the Panama Canal (16) or by the insidious tax across every economic sector in the most highly affected areas (17,18). This amounts to $12 billion lost annually in Africa (19), where affected nations suffer more than a full-percentage point reduction in yearly per capita economic growth due to this disease (20,21). As a further insult, malaria kills hundreds of thousands of children every year (22–24), beginning at the youngest ages (25), and those who survive may experience neurological sequelae (26,27) and reduced educational success (28–31). Malaria impacts all facets of society and is rightly considered a scourge of humankind.

a Human malaria infection is caused by one of four species of Plasmodium: P. falciparum, P. vivax, P. malariae, or P. ovale. In addition, the simian parasite, P. knowlesi, occurs as a zoonotic infection in parts of Malaysia (4,5).

The potential benefit of a vaccine is matched only by the difficulties facing those trying to develop it (32). Expertly adapted to surviving long term in its hostb, the parasite evades or manipulates to its advantage the host immune response, the very tool that scientists must use to suppress or eliminate the infection (36–44). As a pathogen, it is exceedingly complex, with a multistage life cycle characterized by stage-specific expression of proteins (45) and a bewildering choice of 5300 genes that could be selected as components of a vaccine (46). This diversity is augmented by the often extensive polymorphisms of candidate antigens (47), which may induce allelespecific immune responses unable to provide cross-protection against the diverse genotypes encountered in the field (48). After decades of development, the leading vaccine candidate, RTS,S, projected for licensure in the next four to five years (49), appears unlikely to significantly reduce the prevalence of infection, although it stands a reasonable chance to offer a degree of protection against clinical illness (50). Perseverance and possibly a measure of good fortune will be needed to realize the dream shared by so many to provide this one critical tool in the struggle for world health.

Life Cycle Just moments of probing by the feeding mosquito are required for the infectious stage, the 7- to 10-mm spindle-shaped sporozoite, to be deposited with salivary gland secretions into the skin (51). The motile parasite immediately moves toward capillaries using a molecularly driven gliding mechanism (52), enters the lumen of a vessel, and is swept by the blood stream to the liver; there, cued by unknown signals, it crosses

b The malaria species with the shortest duration infection is P.’falciparum, which according to one estimate, may persist up to three years if left untreated (33), although it is likely that many infections are cleared over shorter periods (34,35).

Chapter 69: Malaria Vaccines in Clinical Development

Figure 1 Life cycle of Plasmodium falciparum.

the endothelium to invade hepatocytes (53,54). The process of invasion unfolds as a series of remarkable behaviors that include passage through Kuppfer cells (54,55), followed by the penetration and destruction of multiple hepatocytes (56). This preliminary encounter leaves a pathway of cellular debris, laced with the parasite’s surface-coat (57), the circumsporozoite protein (CSP). Although a local inflammatory response develops, it is largely ineffectual and the sporozoite is able to develop without hindrance (Fig. 1). After its destructive migration, the parasite undergoes an invagination-mediated entry into an intact hepatocyte, assuming intracellular residence encased in a parasitophorous vacuole derived from the host cell membrane (58). This selected host cell increases many fold in size during the ensuing days, transforming through the asexual multiplication of the parasite into a nest of tens of thousands of progeny that will be released back into the vasculature. The duration of development in the liver ranges from a minimum of five and a half days for P. falciparum to multiple weeks or months for P. vivax and P. ovale. The hepatic stages of these two latter species are characterized by a subpopulation of ‘‘sleeping forms’’ or hypnozoites that develop slowly, enabling the reinitiation of blood-stage infection at time points distant to the primary infection (59,60)c. Malaria relapse presumably represents a mechanism to prolong transmission, and in the case of P. vivax adapted to winter c Remarkably, a relapse of P. ovale has been recorded four years after primary infection (61).

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climates, allows the parasite to survive the seasonal absence of the vector. During hepatic development, as with the sporozoite’s journey from the site of inoculation to the liver, the human host experiences no symptoms. The hepatocyte represents a haven protecting against exposure to an array of host defenses including antibodies and complement, although there is evidence that the immune system can nevertheless target hepatic stages during the course of natural infection (62–66). The cell membrane serves as the interface for an influx of nutrients enabling parasite growth and reproduction, as the protozoa undergoes schizogony (multiplication of nuclei and other organelles followed by cytokinesis) resulting in a 30,000- to 40,000-fold increase in cell number (67). This culminates in the budding off from the host cell into the blood stream of membrane-bound packets (called merosomes or extrusomes) containing thousands of merozoites (68), initiating a new cycle of invasion and destruction in the blood that after a week results in an additional million-fold increase in parasite numbersd. The rupture of parasitized erythrocytes and release of merozoite progeny causes the classic clinical syndrome of malaria: paroxysms of headache, violent shaking chills, high fevers and prostration. To the extent that blood-stage replication is synchronized into discrete cohorts of parasites, these paroxysms occur every two to three days, depending on the speciese, with relatively asymptomatic periods in between. In a minority of those infected with the most dangerous of the four species, P. falciparum, sequestering of parasitized erythrocytes in the capillaries and postcapillary venules during the latter stages of each cycle leads to obstruction of vascular flow and a cascade of metabolic derangements resulting in hypoxia and cytokine release and subsequent end organ damage (70). Depending on where sequestration occurs, this may involve deteriorating consciousness, pulmonary capillary leakage, renal failure, hepatic disturbance or dysfunction of other organs (71). Individuals with severe malaria may experience seizures, brain edema, respiratory failure, and a relentless life-threatening metabolic acidosis (72,73). The likelihood of death from severe malaria is high without emergent treatment (74,75). Control of the initial burst of blood-stage multiplication appears to depend on the induction of antibody-mediated immune responses specific to immunodominant surfaceexpressed antigens. The best studied is the P. falciparum erythrocyte membrane protein 1 (PfEMP1) (76), which exists as fifty to sixty antigenically distinct forms, each individually encoded by the genome and expressed sequentially by the parasite within the erythrocyte and displayed on the surface, with the allelic diversity greatly expanded if parasite populations are sampled across the globe (77). Repeated induction of allelespecific immune responses (78) and associated antigenic switching by the parasite lead to sequential waves (recrudescences) of parasitemia and recurrent bouts of clinical illness (79,80). PfEMP1 has specific endothelial binding characteristics that in a given host likely determine the location of the vascular sequestration (81). Antibodies directed to this molecule can reduce endothelial binding (82) and thereby enhance splenic clearance of parasitized erythrocytes (83). The absence of any Dondorp et al. estimated average parasite burden to be 7
1011 parasites in hospitalized patients in Thailand (69). d

e P. falciparum, P. vivax, and P. ovale exhibit a 48-hour cycle, while P. malariae exhibits a 72-hour cycle.

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significant immunity to PfEMP1 and other surface-expressed variant antigens may partly explain why P. falciparum malaria is most severe in naı¨ve individuals, including infants and young children living in endemic areas and travelers of any age. P. falciparum malaria is also more severe in women during their first pregnancy (84), when the placenta serves as a novel sequestration site targeted by parasites expressing PfEMP1 with binding motifs such as var2CSA that recognize receptors in the placenta (85). The allelic switching characteristic of PfEMP1 and possibly other parasite proteins expressed on the erythrocyte surface prolongs the duration of infection but also suggests a target for vaccine-induced immunity, if conserved epitopes can be identified that allow for cross-protection among the variant alleles. After several days of asexual reproduction, gametocytes appear in the peripheral circulation, although the mechanism whereby this sexual stage emerges is not well understood (86). Mature male and female forms (micro and macrogametocytes, respectively) circulate passively in the periphery of the vasculature until imbibed during the blood meal of a female Anopheles mosquito. In the mosquito gut, the sexual forms further develop and unite, in a process akin to fertilization of sperm and egg, creating a briefly diploid organism that encysts on the basal lamina of the midgut outer cell wall. These oocysts develop over several days and release thousands of sporozoites into the insect hemocoel that migrate from the abdomen to the salivary glands. Approximately 14 days following the blood meal, depending on the ambient temperature, sporozoites become infectious and may be transmitted to the next host (87–89).

Targeting the Parasite Strategies for vaccine development generally target specific stages of the parasite life cycle. This is necessary because the expression of many candidate antigens is stage specific, reflecting their underlying biological role. For example, the CSP is expressed on the surface of sporozoites and during the first few days of development within the hepatocyte, but is not expressed during the later hepatic stages or in the blood (90).

Thus a highly effective vaccine based on CSP will not directly impact asexual blood-stage growth, meaning that the vaccine must be 100% effective at killing the pre-erythrocytic stages to prevent clinical malaria. Because this may be hard to achieve, many envision a ‘‘two-tiered’’ vaccine defense that includes antigens from the sporozoite and liver stages, in an attempt to sterilize the infection during its asymptomatic early phase, and antigens from the blood stage, in an attempt to quell asexual growth and the associated clinical syndrome (32) (Fig. 2). The sexual stages represent an additional target (91). Antibodies recognizing sexual-stage antigens that are newly expressed (or newly exposed) in the mosquito midgut can be induced in the human host and passively transferred to the mosquito during the blood meal to disrupt subsequent parasite development (see section on transmission-blocking vaccines). The parasite life cycle also impacts vaccine design by determining the immune responses required for protection at each stage. Sporozoites and merozoites exist free in the plasma, and should be vulnerable to antibody responses. Antibodies may also bind to parasite antigens expressed on the surface of infected cells, such as erythrocytes, and mediate clearance in the spleen or other antibody-dependent mechanism. Hepatic stages, on the other hand, likely require T cell–mediated immune responses for clearance (92). Antigen-specific CD8þ T cells can recognize parasite antigens expressed on the surface of infected hepatocytes, and either kill the infected cells directly [cytotoxic T lymphocyte responses (CTLs)] or kill the parasite within (through an interferon-g-mediated mechanism involving the release of nitric oxide). There is evidence that all of these immune responses play a significant role in protection against malaria (93). Thus the platform selected for the delivery of antigens is a key element in determining protective efficacy. At this point, no platforms have been identified that simultaneously and maximally engage all arms of the immune system believed to be important to protection against malaria.

Public Health Objectives Although the ideal malaria vaccine should provide long-term, sterile protection against infection in all vaccine recipients, the

Figure 2 ‘‘Two-tiered’’ vaccine defense to prevent clinical malaria. In the absence of a vaccine (solid line A), parasites inoculated via mosquito bite develop in the liver and then are released into the blood stream, where they multiply logarithmically for several days until numbers are sufficient to induce symptoms (clinical threshold). A pre-erythrocytic vaccine destroys parasites during the sporozoite or liver stages, ideally eliminating all parasites thereby preventing blood-stage infection (descending dotted line B). However, should some parasites escape so that merozoites are released into the blood stream, the immune system will have more time to respond prior to the clinical threshold, restricting parasite density and ameliorating clinical expression (ascending dotted line B ). A blood-stage vaccine, acting alone, could in theory prevent parasite densities from crossing the clinical threshold (upper dashed line C ). A combined stage vaccine would give the best result (lower dashed line C ).

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difficulty of achieving this aim has led to the concept of developing vaccines designed to protect discrete populations. For example, a vaccine targeting pre-erythrocytic antigens might succeed in preventing blood-stage infection altogether in travelers, who generally experience less intense transmission than those living permanently in endemic areas. On the other hand, a vaccine targeting blood-stage antigens, to reduce parasite density and associated morbidity, might be more appropriate for infants and children living in endemic areas under conditions of intense transmission, enabling the acquisition of clinical immunity while minimizing the severity of the first several infections. The feasibility of developing malaria vaccines of these types is demonstrated by two models of immunity in humans, with each of these models indicating a particular public health strategy. The first model is that of immunization with irradiated sporozoites. It was discovered that the administration of infectious sporozoites attenuated via g irradiation could lead to sterile protection against subsequent challenge with intact sporozoites in rodents, nonhuman primates, and humans (94,95). In animal models of irradiated sporozoites, the vaccine is delivered intravenously, which is not practical for human administration, while in the human model, the sporozoites are delivered via the bites of irradiated, sporozoite-laden mosquitoes, with greater than 1000 infectious bites, administered over the course of multiple immunization sessions, required to provide >90% sterile protection. As mosquito bite delivery is equally impractical as intravenous delivery, and living sporozoites cannot be cultured and cannot be stored except in liquid nitrogen, this model has been deemed impractical for development as a licensable productf. However, it does provide proof of principle for developing a highly efficacious vaccine targeting pre-erythrocytic stage parasites. The task for vaccinologists is to identify the protective antigens and deliver them in such a way as to induce the protective immune response. Killing of pre-erythrocytic stages appears to be mediated by CD8þ T cells recognizing parasite antigens expressed on the surface of infected hepatocytes, with CD4þ T cells also contributing (92). On the basis of this model, vaccine developers envisage developing an ‘‘anti-infection’’ vaccine, with viral vectors or other genetic vaccine strategies selected as the preferred platform because of their superiority to recombinant protein for inducing cell-mediated immunity. This type of vaccine would be designed for military personnel or other travelers to endemic areas for whom transmission is generally neither intense nor prolonged, with a goal to provide complete protection against malaria for a relatively short period of time and under conditions of moderate transmission. The second model is naturally acquired immunity (NAI): over many years of exposure, children living in endemic areas develop a degree of parasitological and clinical immunity to malaria so that the density of parasites in the blood and the severity and frequency of clinical episodes diminish (96). In areas of high transmission, NAI is well established by mid childhood, and older children and adults are clinically protected. NAI is thought to be mediated by antibodies to an array of blood-stage proteins expressed by merozoites or on the

f Very recently, this position has been reconsidered, and there are now active efforts to develop bioengineering processes permitting the delivery of sterile, irradiated sporozoite by needle and syringe (see chap. 71).

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surface of infected erythrocytes (97), as demonstrated by the reduction in parasites counts and clinical symptoms following the infusion of immunoglobulin purified from the blood of semi-immune African adults (98,99). Some of these antibodies may neutralize malarial toxins released by the parasite or on rupture of infected erythrocytes (100). Possibly on the basis of a similar mechanism, women become increasingly resistant to severe disease during pregnancy with increasing parity; it is hypothesized that they gradually develop cross-neutralizing antibodies that inhibit the binding of parasitized erythrocytes to the placenta (101,102). Although NAI is never sterilizing (9), and diminishes gradually if the individual lives for a period of time in a nonendemic area (103), it does prove that an antidisease vaccine is feasible. As with the irradiated sporozoite model, the task remains to identify the target antigens and formulate them into a vaccine. This has become the objective for funding agencies wishing to impact malaria morbidity and mortality in residents of endemic areas, especially where transmission is intense and prolonged. In an attempt to mimic NAI an ‘‘anti-disease’’ vaccine is thus envisioned to be composed of blood-stage recombinant proteins formulated to induce strong antibody responses, possibly delivered in prime-boost combination with other vaccine platforms expressing the same antigens. Although the distinction between these two vaccine types has been important in determining approaches to vaccine development—pre-erythrocytic stage antigens/gene-based platforms/cellular response-inducing/to prevent infection, and blood-stage antigen/protein-based platforms/antibodyinducing/to prevent disease—experimental data from field trials has now challenged these assumptions. For example, the RTS,S vaccine, based on the pre-erythrocytic protein, CSP, may be inducing an anti-disease immune response even though it does not target blood-stage antigens. As these distinctions have become blurred by the data emerging from vaccine field trials, vaccine developers are questioning their assumptions regarding how malaria vaccines should be designed. The discussion of vaccine delivery systems starts in this chapter with the most straightforward approach—the recombinant protein and synthetic peptide platforms formulated with adjuvants. The following two chapters will discuss genetic approaches (chap. 70) and attenuated organism (chap. 71) vaccines.

RECOMBINANT PROTEIN AND PEPTIDE SUBUNIT VACCINES Antigen Expression, Production, and Formulation In developing subunit vaccines, the antigens or critical epitopes must be expressed and formulated with a conformation that elicits a potent and protective immune response recognizing native parasites. Expression of malaria proteins in standard recombinant systems has been problematic with the need for synthetic genes, control of aberrant glycosylation patterns and protein folding. Characteristic of many malarial antigens is their complex disulfide bond-dependent structures making these molecules particularly difficult to produce. Problems with folding and/or disulfide bond formation are exacerbated when expressing proteins with complex secondary structures and multiple disulfide bonds. A variety of expression systems have been employed including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, insect cells, mammalian cells and plant cells. Both native and

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synthetic genes (codons optimized to the expression system being used or codon harmonized reflecting controlled ribosomal pausing) have been used. The synthetic gene approach, which facilitates the correct folding of domains, in general results in better expression than native sequences; this is particularly true of the AT-rich P. falciparum genes. Additionally, full-length antigens, fragments consisting of single and multiple domains, antigens derived from different malarial species, clones and chimeric molecules have been expressed. Attempts have been made to identify regions of the candidate proteins that can be folded either in vivo (e.g., P. pastoris or mammalian systems) or in vitro (e.g., E. coli) to generate molecules structurally indistinguishable from their native counterparts. The subsequent characterization and stability of both the antigens and their formulations are also critical.

Adjuvants and Their Use in Malaria Vaccine Development The malaria parasite has existed for thousands of years in man and other animals. As a consequence of this coexistence the parasite has evolved mechanisms to evade the natural immune responses of its host. These include antigenic polymorphisms and many adaptations that interfere with the development of strong, effective immune responses to a given malaria protein (104). Many malaria antigens appear to be intrinsically poor immunogens (105,106). For example, the presence of immunodominant repeat sequences, characteristic of many malaria proteins, may divert immune responses away from other, potentially protective epitopes located in adjacent regions of the molecule (107), and vaccines including these repeat regions may accentuate the immunodominance of repeat sequences relative to responses induced by natural infection (108). As another example, variant sequences have been identified in key epitopes (altered peptide ligands) that prime immune responses with ineffective cytolytic or lymphokine-secreting ability (immune interference) (109). In other cases, parasite proteins may contain a paucity of epitopes binding strongly to human major histocompatability complex (MCH) molecules, which could explain why certain individuals may not respond at all to a given vaccine, even when multiple antigens are included (110). Often for unclear reasons, vaccines tested in humans are weakly immunogenic, and functional immunity easily achieved when the same immunogens are tested in animals is not induced in the clinic (111).

With the advances in our understanding of immune mechanisms, developers of malarial vaccines are attempting to counteract these adaptations by delivering recombinant parasite antigens with compounds that have a stimulatory effect on the immune system. A fine balance has to be established, however, between too much immune stimulation that might result in undesirable adverse events and activation of the immune system to produce the desired parasite-destructive immune responses. Currently, there are several immunostimulatory compounds, or adjuvants, being evaluated in clinical trials. These can be broadly classified into two categories: (1) depot effect adjuvants and (2) cellular activation adjuvants. The depot or repository adjuvants act by counteracting rapid dispersal of antigen upon injection. The most common adjuvants of this type used in man are aluminum salts (commonly referred to as alum) including aluminum hydroxide (alhydrogel) and aluminum phosphate. These are hydrophilic suspensions on which the antigen is adsorbed. Alum was first used as an adjuvant more than 80 years ago and is still the only preparation approved by the FDA for human vaccines. Other repository adjuvants, still in the licensing phase, include various oil and water emulsions, liposomes and immune-stimulating complexes called ISCOMs (which are comprised of saponin, cholesterol, and phospholipid). The immunogen is emulsified in these mixtures before injection and released slowly from the suspension in the tissues. Cellular activation adjuvants, on the other hand, work by either stimulating macrophages to increase the concentration of processed antigen on their surface for better presentation to lymphocytes or by stimulating the formation of modulating cytokines. This activation of macrophages is achieved by compounds in the emulsions such as surfactants or bacterial products. Some adjuvants incorporate a chemical variant of endotoxin called monophosphoryl lipid A (MPL) or a modified muramyl dipeptide (MDP) or other ‘‘detoxified’’ cell wall constituents of bacteria (112). The identification of effective adjuvants for use in malaria vaccines has not been easy (Table 1), and can be illustrated by the development of three vaccines: the RTS,S vaccine by GlaxoSmithKline (GSK) Biologics and the Walter Reed Army Institute of Research (WRAIR) (113) (49) (114), the ICC-1132 vaccine by New York University (NYU), Oxford University and Apovia, Inc. (115,116) and the AMA1-C1 vaccine by the U.S. National Institutes of Health (NIH) (117). In each case, the antigen has been tested in a variety of

Table 1 Adjuvants in Clinical Development with Malarial Antigens Adjuvant category

Adjuvant

Adjuvant composition

Antigen formulations

Depot/repository

Aluminum salts

Aluminum hydroxide (alhydrogel), aluminum phosphate Liposomes, MPL, and QS21 Oil-in-water emulsion, composed of MPL and QS21 Oil-in-water emulsion, composed of MPL and QS21 Water-in-oil emulsion, composed of squalene and highly purified mannide monooleate Water-in-oil emulsion, composed of paraffin oil and highly purified mannide monooleate CPG 7909

AMA1-C1, MSP142-C1, MSP3, GMZ2, LSA-3, SERA, EBA175 RTS,S, LSA1, FMP1, FMP2.1 RTS,S, LSA1, FMP1, FMP2.1, FMP10

AS01B AS02A AS02D ISA 720

ISA 51

Cellular activation

TLR agonists

RTS,S AMA1, AMA1-C1, CP2.9, ICC-1132, LSA3, MSP-3 Pfs25, Pvs28

AMA1-C1, MSP142-C1, BSAM-2

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adjuvant formulations, which often have yielded significantly different results. In 1984, GSK in collaboration with WRAIR developed the use of hepatitis B surface antigen (HBsAg) as a carrier matrix for P. falciparum CSP (118). A fusion protein named ‘‘RTS,S’’ to indicate the presence of the CSP repeat region (R), T-cell epitopes (T), and the HBsAg was combined with unfused HBsAg and formulated with various GSK preparatory adjuvant systems (AS) (119,120). When formulated with alum it was safe but no protective immune responses were generated. However, when formulated with alum and MPL in combination, two of eight vaccinees were protected. A series of further experiments in rhesus monkeys revealed that one formulation, consisting of RTS,S in an oil-in-water emulsion with MPL and QS21 (known as AS02A) provided high antibody and delayed-type hypersensitivity (DTH) responses. Initial clinical trials at WRAIR with the RTS,S/AS02A formulation given on a zero-, one-, and six-month schedule protected an unprecedented six of seven volunteers (49). Subsequent trials with a shortened zero-, one-, and two-month schedule have achieved levels of success in the range of 30% to 50% protection (114). This illustrates how adjusting the adjuvant formulation for RTS,S converted a completely non-protective immunogen into a strongly protective vaccine. Another protein malarial vaccine, ICC-1132, was developed by NYU and tested by Apovia, Inc. in collaboration with Oxford University. It was a recombinant virus-like particle (VLP) comprising a modified hepatitis B core protein with a Bcell (NANP) and two T-cell epitopes of the P. falciparum CSP. Initial preclinical studies in mice and monkeys when formulated with alum proved the vaccine to be safe and immunogenic (121). The first phase 1 trial using alhydrogel demonstrated CSP-specific repeat antibodies and CSP-specific IFN-g-producing T cells when three doses of 50 mg were administered (122). Preclinical studies in mice and monkeys (121) showed that formulation in Montanide ISA 720 was more immunogenic than alum formulations but also more reactogenic, with some monkeys developing sterile abscesses at the site of injection, particularly after booster immunizations (123). A phase 1 trial of ICC-1132 was designed to assess the safety of a single immunization of 5, 20 or 50 mg ICC-1132 emulsified in ISA 720 (115). The majority of the volunteers in the two higher doses developed CSP repeat-specific antibodies as well CSP-specific cellular cytokine responses. It was not possible to determine whether CD4þ and/or CD8þ T cells were producing IFN-g because of the limited number of PBMC available. A follow on phase 1/2a trial of a single 50mg dose in ISA 720 was safe but induced low anti-repeat antibodies and only modest T-cell responses. There was no evidence of protection against experimental sporozoite challenge. Thus assessing multiple formulations of ICC-1132 did not achieve a protective response and further evaluation of the ICC-1132 vaccine was halted. A vaccine against multiple alleles of the apical membrane antigen 1 (AMA1) from two clones of P. falciparum (3D7 and FVO) termed AMA1-C1 has been developed by the NIH. It was originally formulated on alhydrogel but, while safe, did not induce high-titer antibodies in most vaccinees participating in a phase 1 trial (124). Subsequently, the addition of the immunostimulatory oligodeoxynucleotide (CPG 7909) to the alhydrogel formulated proteins substantially increased the antibody titers in mice, rats and guinea pigs in preclinical trials (117). CPG 7909 is known to prime a strong Th1 response through its

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interaction with Toll-like receptor 9 found in B cells, predominantly memory cells, and plasmacytoid dendritic cells (pDC) (125,126). The uptake of CPG 7909 leads to the activation of cell signaling pathways that culminate in the secretion of Th1 chemokines and cytokines such as IFN-g that support the production of IgG2a antibodies and the development of antigen-specific CTLs. Further tests of the AMA1-C1/alhydrogel þ CPG 7909 vaccine have been carried out in phase 1 trials of adults in the United States and Mali, West Africa. Increased antibody levels have been observed relative to AMA1-C1/ alhydrogel (127–129). The vaccine is now undergoing phase 1 testing in Malian children and will be considered for phase 2b testing to access efficacy against clinical malaria in the field. These three vaccine development histories stress the need for extensive preclinical development followed by clinical testing of new adjuvant combinations, including diligent assessments of adjuvant and antigen dose as well as the timing and number of administrations, to achieve the desired balance between too much reactogenicity and too little immunogenicity. Although partial protection was achieved with RTS,S after testing several different adjuvants, the failure to protect with ICC-1132 despite multiple formulations implies that the development of novel adjuvant systems may be an important prerequisite to achieving sufficient immunogenicity for many malarial antigens.

Pre-erythrocytic Malaria Vaccines Introduction Pre-erythrocytic malaria vaccines target those stages of the parasite development that occur before the release of hepatocytic merozoites into the blood stream. These include the sporozoites and the hepatocytic stages. The mode of action whereby current vaccines that target pre-erythrocytic stage proteins induce protection in humans is still controversial. For example, it is known from animal models that antibody against the repeat region of CSP will completely protect animals from malaria following sporozoite challenge (130). However, in human vaccine trials, some individuals have been protected though they developed only low to moderate antibodies (49,131), and there is good evidence that CD4þ and/or CD8þ T-cell responses play a role in protective immunity (132). Sun et al. have described a correlation between high IFN-g production and protection in volunteers in an RTS,S vaccine trial yet there were also individuals in the same study who were not high IFN-g producers but were protected and individuals who had high IFN-g production but were not protected (133). Animal models generally illustrate the complexity of potentially protective immune responses rather than providing definitive information regarding what responses will be most protective in humans (134). Clearly, multiple immune mechanisms act on the pre-erythrocytic stage parasite and the building of an effective vaccine to stop malaria will likely require the stimulation of a multifactorial immune response against a variety of pre-erythrocytic stage antigens, and may require targeting multiple stages as well. The purpose of a vaccine directed against the pre-erythrocytic stages of malaria is to completely prevent blood-stage infection and thus clinical disease. Because there are no observable clinical symptoms caused by sporozoites or developing hepatic schizonts if the parasite can be killed before it enters the blood stream, any manifestations of disease can be entirely prevented.

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Development of Pre-erythrocytic Malaria Vaccines One of the most significant tools available to malaria vaccine developers is the ability to challenge individuals with malaria parasites in a controlled, reproducible manner. In 1985, a system was established for using membrane-fed anopheline mosquitoes carrying P. falciparum to infect volunteers (135). Seed lots of cloned parasites with known sensitivities to antimalarial compounds could be made and a repeatable infectivity model for evaluating vaccine or drug efficacy could be established. A retrospective analysis of the first 118 volunteers participating in studies using the 3D7 clone of a P. falciparum isolate conducted between 1985 and 1992 showed the method to be a reliable, safe and well-tolerated experimental model (136). An analysis of more recent data has confirmed the safety and reliability of the challenge model (132). To date, over one thousand three hundred people have been challenged by this method and no recrudescence of parasitemia has occurred after treatment, eliminating the risk of delayed clinical illness or secondary transmission after the trial. The more recent introduction of quantitative real-time PCR permits two- to four-dayearlier detection of parasitemia (shortening the pre-patent period) and allows the estimation of critical parameters in the parasite life cycle using a statistical model, including backcalculating the number of infected liver cells and estimating the rate of parasite multiplication of blood stages (137,138). Pre-erythrocytic Malarial Vaccines in Clinical Development Four pre-erythrocytic proteins, which have reached clinical testing, will be discussed: CSP and thrombospondin-related adhesive protein (TRAP or SSP2) from the sporozoite stage, and liver-stage antigen-1 (LSA1) and liver-stage antigen-3 (LSA3) from the liver stage. Circumsporozoite protein. CSP is a leading candidate for pre-erythrocytic-stage vaccines. The protein is encoded by a single-copy gene and covers the surface of sporozoites (139). CSP from Plasmodium species display common structural features, including an N-terminal signal peptide, a C-terminal glycosylphosphatidylinositol (GPI) anchor that links the protein to the sporozoite surface, and a central domain composed mostly of amino acid repeats. The repeat region is immunodominant and is a target for neutralizing antibodies against sporozoites (140). Flanking the central repeats, all CSPs contain highly conserved domains designated as Region I, Region IIplus, and Region III. Region I contains a pentapeptide, KLKQP, and is involved in attachment of sporozoites to mosquito salivary gland and liver tissue (141,142). Region II-plus consists of a pair of cysteines and a six amino acid consensus sequence and is embedded at the proximal end of the thrombospondin domain. The domain contains CD4þ and CD8þ T-cell epitopes, and is involved in adhesion to and invasion of mosquito salivary gland and human liver tissues (143). Region III is predicted to form an amphipathic a-helix and may provide a proper framework for the neighboring Region II-plus adhesion motif (144). While there have been many CSP-based vaccines tested, only the RTS,S recombinant protein/adjuvant combination has proven to protect people against sporozoite challenge in clinical settings and the field. RTS,S includes only a portion of the CSP molecule, and >80% of the antigenic component of the vaccine is actually HBsAg, which promotes spontaneously assembly into VLPs. To achieve protection, RTS,S is formulated by GSKBio in the adjuvant systems AS02A, AS01B, or AS02D, and consistently protects 40% to 50% of immunized volunteers

against experimental sporozoite challenge with the homologous parasite strain (114,145). Results of meta-analyses by Graves and Gelband (146) of nine safety and efficacy trials, and two safety trials, with over 3000 participants of semiimmune children, of the RTS,S vaccine, showed that it reduced clinical episodes of malaria by 26% and severe malaria by 58% for up to 18 months. Prevalence of parasitemia was also reduced by 26% at six months after immunization. RTS,S also reduced clinical malaria episodes by 63% in semi-immune adult men in the second year of follow-up after a booster dose. Most recently, in a study not included in the metaanalyses, RTS,S formulated in AS02D reduced the rate of new onset parasitemia in infants over a six-month observation period by 66% (95% CI, 43–80%) (147). At the same time, other CSPbased vaccines tested in the field have shown no evidence for a protective effect, including the CS-NANP vaccine [307 participants, three trials (148–150)] and the CS102 peptide vaccine [14 participants, one trial (151)]. Likewise, trials of vaccines containing a second sporozoite antigen, the thrombospondin-related adhesive protein (TRAP or SSP2), also failed to protect in field trials [the ME-TRAP vaccine, 777 participants, two trials (152,153)]. The analysis by Graves and Gelband concluded that the RTS,S vaccine was effective in preventing a significant number of clinical malaria episodes, including good protection against severe malaria in children for 18 months with no severe adverse events attributable to the vaccine (146). While the report recommended progression of the RTS,S vaccine toward licensing, it stressed the need to increase its efficacy. The report did not identify evidence for supporting additional development of other vaccines included in the review, and recommended further research on other CSP-based vaccines. In an early attempt to increase the efficacy of the RTS,S vaccine, GSK and WRAIR conducted a combination trial administering both RTS,S and TRAP proteins simultaneously. Though the number of volunteers was small, there were no protected individuals and it appeared that the TRAP protein interfered with the RTS,S vaccine’s ability to induce a protective immune response (113). The prime-boost combination of RTS,S with other vaccine platforms, such as adenovirus vectored vaccines, may be profitable (154). Thrombospondin-related adhesive protein. TRAP is a P. falciparum 90-kilodalton (kDa) protein expressed in both the sporozoite and asexual erythrocytic stages. TRAP is localized to the microneme and cell surface of mature sporozoites and has been considered to play a critical role in gliding motility and in the recognition and/or invasion of hepatocytes. Cytotoxic CD8þ T cells recognizing TRAP have been identified in humans immunized with irradiated P. falciparum sporozoites and protected against experimental sporozoite challenge (155,156). Furthermore, antibodies against TRAP have been shown to block the sporozoite invasion into hepatocytes in vitro (157). Naturally acquired antibodies against TRAP in combination with high antibody titers to CSP and LSA1 correlate inversely with the malaria parasite densities among children in a hyperendemic area (66,158). Murine studies support the role of TRAP as a protective pre-erythrocytic antigen. P. yoelii SSP2 (the murine parasite equivalent of TRAP) has been shown to be the target of protective CD8þ CTL that eliminate P. yoelii–infected hepatocytes in mice (159), and immunization with a synthetic branched-chain peptide including four copies of a PySSP2 sequence, NPNEPS, formulated in adjuvant, protected A/J, but not BALB/c or C57BL/6 mice (160). In the first study,

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transfer of T lymphocyte–enriched immune splenocytes recognizing TRAP protected naive mice (159), while in the second, in vivo depletion of CD4þ T cells eliminated vaccine-induced protection and in vivo treatment with anti-IFN-g reversed vaccine-induced activity against infected hepatocytes (160). As mentioned above, immunization of humans with a recombinant protein formulation of TRAP failed to protect (113). However, given the protection observed with the PySSP2 in the murine model and the ability of immune responses targeting the PfTRAP to reduce or block sporozoite infection of hepatocytes, it is believed that presentation of the TRAP molecule in a context that induces high antibody titers coupled to potent CD8þ T cells should constitute an effective vaccine. Indeed, studies of TRAP delivered as a heterologous regimens of DNA and recombinant poxviruses demonstrated protection against experimental sporozoite challenge in nonhuman primates (161) and in humans (162,163). Disappointingly, however, these prime-boost genetic vaccines based on TRAP failed to protect in two field trials (152,153) (see chap. 70). Liver-stage antigens (LSA1 and LSA3). Substantial epidemiological evidence has suggested that individuals who were naturally protected from malaria recognized determinants expressed on the protein LSA1 (164). LSA1 as expressed in native form by P. falciparum is a large 230-kDa MW protein having conserved N- and C-terminal regions flanking a region containing approximately 86 repeats of 17 amino acids each. An LSA1-based protein vaccine, LSA-NRC, containing the entire amino and carboxy-terminal regions of LSA1 flanking two repeats (165) was emulsified in either AS01B or AS02A adjuvant and administered to volunteers in a combined phase 1/2a trial. Despite the induction of high LSA1-specific antibody and IFN-g producing T-cell responses, none of the vaccinees were protected or showed any significant delay in onset of parasitemia following sporozoite challenge (Cummings, manuscript in preparation). LSA3 is a pre-erythrocytic antigen found in infected liver cells but also in erythrocytic stages. It is an antigen that contains highly conserved regions as well as highly variable regions. Long synthetic peptides and recombinant proteins have been made representing the conserved regions, and in limited small trials have protected nonhuman primates, including chimpanzees (166) and Aotus monkeys (167) against P. falciparum sporozoite challenges. A recombinant protein construct is currently being tested in Nijmegen, the Netherlands in a phase 1 conditional phase 2a study (Sauerwein, personal communication). Concluding Remarks The only malaria vaccine that has demonstrated anti-disease effects without necessarily preventing parasitemia is based on a single pre-erythrocytic stage antigen, the CSP. This vaccine, RTS,S, protected 40% to 50% of malaria-naive vaccine recipients tested in the United States against experimental sporozoite challenge (114). While it has less effect on parasitemia in the field, remarkably, it has significantly reduced the frequency of clinical disease for a period of at least 18 months in African children, even though most of these children nevertheless did become parasitemic (168,169) The mechanism for this protective effect is unclear, but may relate to the killing in the liver of more virulent parasite strains (with less virulent strains still getting through), or to a general reduction in the number of parasites entering the blood following liver-stage development. In the latter case, it is argued, a smaller blood inoculum from

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the liver would prolong the time required to the onset of the clinical syndrome, thereby allowing the immune system a greater opportunity to respond effectively. The achievement of clinical protection by a ‘‘leaky’’ pre-erythrocytic stage vaccine was not anticipated, and suggests the need to redefine the classic anti-disease vaccine paradigm. The positive findings associated with RTS,S have emphasized the potential impact of pre-erythrocytic stage antigens on clinical disease. The rationale for a pre-erythrocytic stage approach is strengthened by the finding that reduction in liver-stage burden by other mechanisms, such as insecticidetreated bed nets, also favorably impacts malaria-related morbidity (170,171). It makes sense to attack the early stages of infection, when parasites are present in small numbers (<100), compared with the blood stages, which, if not checked on release from the liver, number in the hundreds of billions.

Blood-Stage Malaria Vaccines Introduction Following release from the liver, merozoites invade red blood cells where multiple cycles of infection result in high-level parasitemia and the many pathological manifestations associated with the disease. Interventions that significantly reduce parasitemia would alleviate morbidity and ultimately reduce mortality in infected individuals. Over time, people living in endemic areas develop natural immunity to P. falciparum as a result of repeated infection, mediated in part by blood-stage parasite-specific antibodies (172) that reduce parasite multiplication rates. Thus, parasite proteins expressed during bloodstage infection have been proposed to be good candidates for inclusion in a vaccine. Candidate antigens for blood-stage vaccine development have been chosen on the basis of such indicators as location on the surface of merozoites or infected erythrocytes, correlation of antibody levels with protection in the field and/or demonstrated protection in animal models. The purpose of an asexual blood-stage vaccine is to elicit immune responses that either destroy the parasite in the blood stream or inhibit the parasite from infecting red blood cells. The net effect is to reduce or prevent the burden of parasites and hence decrease the incidence, severity, or the complications of disease. The initial vaccination could act to prime the immune system for subsequent boosting on exposure to infection or repeat immunization, or it could boost already present, yet weak, natural immunity in young children. The enhancement of naturally induced immune responses could be maintained by subsequent natural infections. Thus, the goal of blood-stage vaccines is not to provide sterile protection against primary infection or disease in malaria-naı¨ve individuals (e.g., travelers), but rather to slow parasite multiplication, thereby limiting morbidity, severe disease, and death in residents of malariaendemic areas, primarily young children and infants. As yet, no blood-stage vaccine has been developed that has achieved any of these favorable outcomes when tested in the field. Development of Blood-Stage Malaria Vaccines The degree of protective immunity in humans has been shown to parallel the level of antibody against asexual blood-stage antigens (173–175), and these levels increase with age. However, the specificity and level of antibody that must be induced to confer protection against clinical disease in malaria is unknown. Immunological correlates will only be obtained

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after successful phase 2 and phase 3 field trials have been undertaken. The most recent data indicate that protection may be mediated by complex patterns of response involving multiple antigens (176,177). Both the apical merozoite antigen 1 (AMA1) and the merozoite surface protein 1 (MSP1) have been implicated in generating antibodies that can block parasite growth within the red blood cell. Rough estimates of the antibody concentrations likely to be useful based on the results of in vitro parasite growth inhibition assays (GIA) have been determined (178). However, a direct correlation between clinical outcomes, specific antibody levels and GIA has yet to be established. Conclusions drawn on GIA results alone (antibody-mediated inhibition of parasite growth in vitro) exclude other possible mechanisms of vaccine-induced parasite clearance (i.e., antibody-dependent, cell-mediated immunity; T cell–dependent killing) and may underestimate the efficacy of a vaccine. Unlike pre-erythrocytic stage vaccines, for which experimental sporozoite challenge provides a good measure of protective efficacy predicting outcome in the field, the only way so far to determine if there is a beneficial effect from a blood-stage vaccine is the outcome of phase 2b studies conducted in endemic settings. This is because volunteers developing malaria following experimental sporozoite challenge must be treated immediately upon the detection of parasitemia, preventing an assessment of the morbidity associated with infection. A number of blood-stage vaccines are thought to function by generating antibody that acts via an antibody-dependent, cell-mediated immune mechanism. These candidates include MSP3, SERA, and GLURP (179–185). MSP1 vaccination may act through a similar mechanism. Several groups have worked to establish assays that can evaluate this biological functioning of specific antibodies (186). However, these techniques have not been easily transferable to other laboratories. The relevance of cell-mediated mechanisms to protect against severe disease has not yet been determined, although cellular protection against experimental blood-stage infection has been demonstrated. In volunteers immunized with repeated cycles of low dose of asexual blood-stage parasites followed by drug cure, protection was observed on subsequent blood-stage parasite challenge in the absence of detectable antibody responses by IFA, implicating a cellular immune mechanism. The hypothesized association between cell-mediated immunity and protection was supported by the induction of a proliferative T-cell response involving CD4þ and CD8þ T cells, a cytokine response consisting of interferon-g but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells and a drop in the number of peripheral natural killer T cells (187). Blood-Stage Malarial Vaccines in Clinical Development A number of P. falciparum merozoite proteins have been identified as promising blood-stage candidate antigens (172) and are currently being evaluated in clinical trials. These include: AMA1, MSP1, MSP2, MSP3, SERA, GLURP, and EBA175. Apical membrane antigen 1. The P. falciparum AMA1 is an unglycosylated, type I integral membrane protein that is synthesized as an 83-kDa precursor in merozoites (188). At about the time of red blood cell invasion, the N-terminus of the 83-kDa precursor is processed to a 66-kDa protein, and AMA1 is translocated to the merozoite cell surface (189). The precise role of AMA1 in the parasite is unknown; however, it plays a critical role in the erythrocyte invasion process across divergent

Plasmodium species. Expression of AMA1 has also been detected in the sporozoite stage of the parasite (45) and suggests an additional role for AMA1 during the liver-stage invasion (190). Targeted disruption of the AMA1 gene in P. falciparum parasites through ‘‘knockout’’ constructs is not possible and indicates a critical role for AMA1 in multiplication of the parasite (191). Therefore, an immune response against AMA1 may have a deleterious effect on liver-stage parasites as well as having a negative impact on blood-stage parasites, thus protecting the host by multiple immune mechanisms. Alignment of published P. falciparum AMA1 gene sequences from different isolates reveals that the AMA1 protein occurs as distinct allelic variants. These point mutations result in at least 68 known polymorphisms in the amino acid sequence (192–194). The three-dimensional distribution of the polymorphic sites shows that all are surface exposed and are exclusively located on one side of the molecule (195,196). Approximately 15% of the AMA1 ectodomain sequence is polymorphic, mostly within regions I and II; region III is the most conserved and predominantly dimorphic (194,195,197). The polymorphisms are very widespread in field isolates and are thought to have emerged as a result of immunological pressure (198,199). AMA1 has been shown in several studies to be a promising malaria vaccine candidate. There is mounting evidence to suggest that this protection is antibody mediated; antibodies raised in various animal species against AMA1 inhibit merozoite growth in vitro (200,201) and in vivo (124,202–208). Human anti-AMA1 antibodies also inhibit merozoite growth in vitro (124,206,207). Protective antibodies react with conformational epitopes with AMA1 (197,209) and these antibodies inhibit parasite growth in vitro in an isolate-specific manner, thus demonstrating that the polymorphisms in AMA1 are not immunologically silent (206). The existence of antibodies and responsive T cells specific to P. falciparum AMA1 in individuals living in endemic areas has been demonstrated (210–212). Thus, the major challenges in the development of an AMA1-based vaccine will be addressing antigenic polymorphism, longevity of the immune response, and vaccination in the context of ongoing infection. AMA1 recombinant protein is currently under clinical development formulated with several different adjuvants. A series of clinical trials have evaluated the vaccine AMA1-C1 formulated on alhydrogel (four trials) and alhydrogel þ CPG 7909 (three trials). AMA1-C1, which contains equal quantities of the FVO and 3D7 forms of the protein expressed from P. pastoris (206), was developed to reduce the effect of antigenic variation. In U.S. adults (124), Malian adults (213) as well as Malian children (214), alhydrogel formulations were safe and immunogenic. However, only moderate levels of parasite growth inhibition were demonstrated by the antibodies produced (124). In a phase 2b study conducted in children living in an endemic area, AMA1-C1/Alhdyrogel showed no effect on time to first parasitemia or parasite density in the initial one-year follow-up period (215). Taken together these results indicate the need to enhance the immunogenicity of the AMA1-C1 vaccine. In parallel to these trials, the addition of the immunostimulator CPG 7909 to the AMA1C1/alhydrogel vaccine has been tested. Adult phase 1 trials in the United States (two trials) and Mali (one trial) have shown the addition of CPG 7909 to AMA1-C1/Alhdyrogel significantly enhanced the antibody response (127–128). This increase in antibody response translated to greater GIA activity (128). In these studies, no significant laboratory abnormalities or

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vaccine-related serious adverse events were observed. AMA1C1/alhydrogel þ CPG 7909 will be evaluated for safety in Malian children. A second AMA1 vaccine, called FMP2.1, has also been evaluated in clinical trials. FMP2.1 was adjuvanted in AS02A, the oil-in-water emulsion containing MPL and QS-21 that was successful with RTS,S. FMP2.1 contains only the 3D7 form of AMA1 expressed in E. coli. Safety, reactogenicity and immunogenicity for FMP2.1/AS02A were evaluated in U.S. adults. The vaccine was shown to elicit potent humoral and Th1-biased cellular immune responses (216). The antibodies were also shown to inhibit parasite growth in vitro. Evaluation of this vaccine in Malian adults demonstrated a good safety and tolerability profile and strong immunogenicity (217). Phase 1 and 2 trials of FMP2.1/ AS02A in children are now ongoing in Bandiagara, Mali. As the AMA1 antigen is also expressed on the sporozoite, FMP2.1 is being assessed for its ability to protect malaria-naive volunteers against experimental sporozoite challenge. Three clinical evaluations of AMA1 formulated in ISA 720 have been conducted. The first study evaluated AMA13D7/ISA 720 compared with ISA 720 alone in Australian adults (218). This formulation was generally well tolerated. Although a number of volunteers in both the vaccine and control groups developed local adverse events, the formulation was not highly immunogenic. This vaccine formulated by homogenization did not contain any excipients or stabilizers and it was determined that the vaccine had lost potency during the trial. The second study was conducted in Europe and compared AMA1-3D7 formulated with alhydrogel, AS02A or ISA 720. Volunteers receiving AMA1-3D7/ISA 720 experienced local adverse events including sterile abscesses in two volunteers; the other adjuvants also induced induration and erythema (219). This vaccine was prepared immediately before use utilizing a dual syringe method. In the final study, Australian volunteers received AMA1-C1/ISA 720 prepared by homogenization with glycine as a stabilizer. This formulation was better tolerated with only a few reported adverse events of erythema and moderate induration (220). The differences in the reactogenicity and immunogenicity of the three AMA1/ISA 720 vaccines can most likely be attributed to the vaccination schedule and the quality of the adjuvant formulations. Post-formulation modifications of proteins and suboptimal particle size of the emulsions may have contributed to the poor immunogenicity or tolerability observed in some studies (221). Merozoite surface protein 1. Native MSP1 is as a ~200 kDa polypeptide that is processed at or just prior to merozoite release from the red blood cell, into smaller fragments that form a noncovalently associated complex (222). The 42-kDa C-terminal fragment of MSP1 (MSP142) is responsible for tethering the complex to the surface of the merozoite (223,224). At the time of merozoite invasion of red blood cells, MSP142 undergoes a secondary processing event and is cleaved into MSP1 33 and MSP1 19 (225). This ‘‘secondary processing’’ of MSP142 is essential for parasite invasion of red blood cells as inhibition of this cleavage inhibits invasion, and methods that prevent invasion also block cleavage (226). The sequence diversity of P. falciparum MSP142 in the parasite population is minimal; MSP142 can be divided into dimorphic and conserved regions (227). The MSP133 domain is dimorphic having two prototype allelic forms, whereas the MSP1 19 domain is highly conserved with six commonly observed amino acid dimorphisms (227–229).

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In Aotus monkeys, protection from high parasitemia correlated with the antibody levels against MSP119 (230). In the mouse challenge model, a functioning intact immune system was required to clear the parasites following vaccination with MSP1 (231). However, passive transfer of antibody to immunocompromised mice was only able to delay the growth of parasites but could not clear infection. In contrast, immunologically intact malaria-naive mice that receive high concentrations of antibody, with time, were able to clear infections presumably because the passively transferred antibodies slowed parasite growth sufficiently for the host to develop an effective immune response. Moreover, MSP1-specific T cells that produced IFN-g also transferred protection. Taken together, these data suggest that MSP1 antibody appears to be important in protection but other immune mechanisms also contribute. Although extensive epidemiological and animal data show that antibodies induced against MSP119 protect against malaria, MSP119 alone as a vaccine does not readily induce parasite-specific humoral responses because it is believed to lack the necessary T-helper epitopes (232,233). For this reason, the larger molecule, MSP142, is a better choice for developing a successful vaccine strategy. The majority of the B-cell epitopes have been localized to the highly conserved region, MSP119 (234), whereas the T-cell epitopes that induce proliferation have been localized to the dimorphic region of the molecule, MSP133 (234,235). In an area where multiple forms of the parasite are present, the development of an appropriate immune response by MSP142 vaccination may require the inclusion of both T-cell epitopes from the dimorphic region as well as the conformation-dependent B epitopes to prime for or boost responses as a result of natural infection. Several MSP142 vaccines are currently under clinical development. A vaccine formulation with the adjuvant AS02A, termed FMP-1, contains the 3D7 form of MSP142 produced as a recombinant protein from E. coli. In the United States, Kenya and Mali adult volunteers this vaccine was found to be safe and immunogenic (236–238). Antibodies from the malaria-naive U.S. volunteers were tested in the GIA assay, but achieved no more than 15% inhibition (236). In Kenyan children, the vaccine was safe and immunogenic (239) but no efficacy against clinical outcomes was observed (240). A vaccine containing the FVO parasite strain of the MSP142 has also been manufactured (FMP-010). Preclinical evaluation of the vaccine in mice, rabbits and primates indicates that it is more immunogenic then the FMP1 formulation and results in higher GIA activity (Angov, personal communication). A phase 1 trial is planned for 2009. There have been two clinical trials using alhydrogel formulated MSP142 recombinant proteins produced from E. coli. The first of these compared MSP142-FVO/alhydrogel and MSP142-3D7/alhydrogel. The second compared alhydrogel formulations of MSP142-C1 (combination of the FVO and 3D7 alleles) vaccine with and without the addition of the immunostimulator, CPG 7909. The MSP142/alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biological effect in vitro (111). The nature of the antibody and T-cell immune responses were qualitatively different. Antibodies generated following vaccination with either MSP142-FVO/alhydrogel or MSP142-3D7/alhydrogel were similar in their ability to recognize the FVO or 3D7 form of the antigen, suggesting limited strain specificity of the antibody response. The majority of the antibodies were reactive with the

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C-terminal region of MSP142. In contrast, cytokine ELISpot analysis of MSP142-specific T-cell responses revealed a strong preference toward the N-terminus of the immunizing antigen with limited activation by the alternate form of MSP142 (241). The addition of CPG 7909 enhanced anti-MSP142 antibody responses following vaccination by 12- to 30-fold two weeks after second immunization when compared with MSP142-C1/ alhydrogel alone (242). A substantial enhancement of specific antibody responses in the CPG 7909 groups correlated with an increased GIA (range 8–32%), but biologically relevant inhibition likely requires much higher levels of anti-MSP142 antibodies. Analysis of T-cell responses in the volunteers receiving MSP142-C1/alhydrogel  CPG 7909 vaccines in these trials is ongoing. Merozoite surface protein 2. MSP2 is an abundant protein on the surface of P. falciparum merozoites as a component of the fibrillar coat. Its specific function is not yet known although the localization of this protein suggests involvement in attachment to the red blood cell membrane. MSP2 appears essential for parasite viability as attempts to knock out the gene have not been successful (243). MSP2 is a 28kDa GPIanchored membrane protein (223,244). MSP2 is a highly polymorphic antigen but like MSP1 it is characterized by two major dimorphic forms (245,246). The protein has a central variable region flanked by conserved N-terminal and C-terminal regions. MSP2 is an intrinsically unstructured protein that polymerizes into amyloid-like fimbrils (Anders, personal communication; 247). Several seroepidemiological studies have shown that the natural anti-MSP2 antibody response is predominantly directed against epitopes in the central variable region (248–250). An association has been shown between antibody responses to MSP2 and resistance to infection (251,252). The preclinical data supporting the candidacy of this antigen are not as extensive as for either AMA1 or MSP1. However, the results of a phase 1/2 clinical trial of the Combination B vaccine (which consists of a mixture of the MSP1, MSP2 and RESA antigens) in Papua New Guinea children suggested that MSP2 has the potential to limit parasite development in vivo. The single type of MSP2 included in Combination B vaccine was underrepresented when parasite isolates identified in vaccine recipients following immunization were analyzed, demonstrating that the vaccine significantly reduced the densities of the homologous parasite strain (253– 255). As a result of this study, MSP2-C1 (a combination of the two dimorphic forms) formulated in ISA 720 has been developed. A phase 1 study in Australian adults has been undertaken; results have not yet been made publicly available. Merozoite surface protein 3. MSP3 is expressed by merozoites and also in late stage schizonts as a 48-kDa protein. Seroepidemiological data have demonstrated an association between anti-MSP3 antibodies and protection (256,257). AntiMSP3 antibodies are predominantly noncytophilic IgM and IgG2 (181) and interact with antibodies to elicit antibodydependent cellular inhibition (ADCI) in vitro (179–182). The conserved C-terminal region of MSP3 contains the epitopes responsible for ADCI. A long synthetic peptide derived from the C-terminal region and containing both B- and T-cell epitopes has been developed for clinical evaluation (258). This long synthetic peptide has been formulated with both aluminum hydroxide and ISA 720 and evaluated in a phase 1 trial. Both formulations were immunogenic with both antibody and cellular responses

detectable (259). Only a subset of the volunteers had antibodies able to mediate killing of parasites by ADCI (260,261). These antibodies could also clear parasitemia when passively transferred to an infected humanized mouse. The aluminum hydroxide formulation of MSP3 has progressed to safety evaluations in children in Burkina Faso and Mali. Erythrocyte-binding antigen 175. EBA175 is a 175-kDa protein, located at the apical end of merozoites, that belongs to a family of erythrocyte-binding proteins with N-terminal cysteinerich regions referred to as Duffy-binding-like domains. The N-terminal region of EBA175 contains a tandem duplication of DBL domains (262,263). Within this domain, a region designated RII binds to sialic acid residues on glycophorin A and mediates parasite entry into red blood cells (264). Although this region appears to be under immune selection (265), antibodies directed against RII have been shown to block parasite invasion of red blood cells in vitro (266,267). A nonglycosylated form of EBA175 RII formulated with the aluminum salt adjuvant Adju-Phos has completed clinical trials. Serine repeat antigen 5. SERA, also known as P126 antigen, is a large protein that is processed into three fragments, 19 kDa, 47 kDa, and 50 kDa (268). It is not expressed on the surface of the parasite but rather accumulates in the parasitophorous vacuoles of trophozoites and schizonts. On the basis of the localization of this protein, it is difficult to explain the mechanism by which anti-SERA antibodies, shown to demonstrate parasite killing in vitro through complementmediated inhibition and ADCI (183,184), would function in vivo. However, significant associations between antibody responses to SERA and lower parasitemia have been shown in cross-sectional studies conducted in the Solomon Islands, Brazil and Uganda (269,270). The N-terminal region of SERA has shown protective efficacy against challenge in nonhuman primates (271,272) and is currently being evaluated in a phase 1 study. Combination blood-stage antigen vaccines. The hypothesis for development of a blood-stage antigen mixture is that vaccines containing multiple antigens to the same life cycle stage would diminish the impact of immunological nonresponsiveness in humans to any one antigen. When the immune response to different antigens is not highly correlated and where each antigen has a modest probability of inducing a protective response, then combining multiple antigens should substantially increase the overall protection in the population. Thus, it is envisaged that different blood-stage candidate antigens could be combined to yield an intervention with greater efficacy than any one antigen alone. In addition to improving immune coverage, combining antigens that are functionally independent should induce immune responses that complement one another in reducing parasite burden. The additive advantage of these immune responses would be limited only by the inability of humans to respond to all antigens within the combination vaccine. The rationale for antigen combination is supported by recent epidemiological data from Kenya that showed the immunity to malaria correlates with elevated antibody levels to multiple blood-stage antigens (177). Major challenges in the development of blood-stage antigen mixture vaccines will include selecting the ideal combination of antigens and assessing the contribution of individual antigens to any protection afforded by the vaccine. As with single antigen vaccines, additional challenges will include measuring the appropriate immune responses, identifying

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criteria for clinical advancement and enhancing the potency and longevity of the immune response. PfCP-2.9 (AMA1 and MSP119 chimera) PfCP-2.9 is a chimeric molecule expressed in P. pastoris that includes the C-terminal region of AMA1 (domain III) and MSP119 from the 3D7 and K1 P. falciparum lines respectively (273). Both components of this chimeric protein are targets of inhibitory antibodies. Sera from animals immunized with PfCP-2.9 inhibited parasite growth in vitro. PfCP-2.9 formulated in ISA 720 has been evaluated in two phase 1 trials. In both trials, the vaccine was well tolerated, immunogenic and recognized proteins on the surface of parasites (274; Malkin, personal communication). The second trial evaluated an optimized formulation and immunization schedule. GMZ2 (GLURP and MSP3 hybrid) GMZ2 is a hybrid molecule including the C-terminus of MSP3 and amino acids 85 to 213 of glutamate-rich protein (GLURP) expressed by Lactococcus lactis (275). GLURP is expressed by the erythrocytic and pre-erythrocytic stages of the parasite, and is a target of protective antibodies from immune adults (276–279). Human anti-GLURP antibodies, in particular IgG1 antibodies raised to amino acids 85 to 213, have been shown to inhibit parasite growth in vitro in the presence of monocytes (185). GMZ2 adjuvanted with aluminum hydroxide is currently undergoing phase 1 trials in Germany and Gabon. Concluding Remarks The identification and generation of the most appropriate Band T-cell immune responses that can curb malaria infection in an endemic setting remain major challenges for vaccine developers. The specificity of the immune response produced as a result of vaccination is important and may be altered by manipulating the formulations (antigens and adjuvants). Additionally, the characteristics of the field site (e.g., entomological inoculation rate, and seasonality of transmission) chosen for testing and the clinical outcomes may impact the results of the trial. Blood-stage proteins have so far fallen short in the promise to induce significant protective responses (280). A blood-stage vaccine including three protein components, called Combination B (composed of MSP1, MSP2 and RESA), failed to prevent clinical disease in children in Papua New Guinea, even though there was a reduction in parasite density in one study group (255), and a leading blood-stage antigen, MSP142, formulated as a recombinant protein in adjuvant, failed to provide protection in Kenyan adults (Angov, personal communication). More recently, a second leading blood-stage antigen, AMA1, despite inducing high antibody titers and CMI responses, failed to induce a significant delay in the onset of parasitemia, as detected by blood smear, in a phase 2a challenge study conducted in the United States (Spring, personal communication). There was a slight delay in parasitemia of vaccinated volunteers compared with nonvaccinated volunteers when monitored by qPCR, but this was not statistically significant. It was not possible to avoid treating these volunteers once parasitemia had developed to see if the vaccine dampened clinical acuity. It could be hypothesized that these antigens are intrinsically unable to induce protective responses; in other words, that they are the wrong choices for inclusion in a vaccine. At this point, the value of blood-stage antigens for achieving antidisease effects remains unproven.

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Transmission-Blocking Malaria Vaccines Introduction Transmission-blocking malaria vaccines (TBMV) are based on sexual- or sporogonic-specific antigens and designed to arrest the development of sporogonic stages inside the mosquito. The specific antibodies generated in the human host are passively ingested together with parasites when mosquitoes take a blood meal and bind to the parasites in the lumen of the mosquito midgut thereby preventing progression of their sporogonic development. Once inside the mosquito midgut, gametocytes rapidly emerge from the intracellular red blood cell environment to prepare for fertilization and are directly exposed to hostile immune components of the ingested blood. The sporogonic cycle may be the most vulnerable part of the life cycle because, at least in the case of some transmission-blocking targets, it appears to have evolved in the absence of immune pressure from the human host, and is therefore an attractive target for interventions. Reduction or absence of the infectious mosquito reservoir will lead to reduction or eradication of malaria in the human population (281). TBMV are fundamentally different from the pre-erythrocytic and blood-stage vaccines previously discussed since they do not provide immediate protection against clinical disease but rather reduce chances to become infected. This differentiation between actual clinical disease and disease risk is due to the special characteristics of the Plasmodium life cycle where separate forms are responsible for disease (pathogenic asexual stages) and transmission (nonpathogenic gametocytes). Asymptomatic parasite carriers are primarily responsible for transmission since that fraction of the population is of larger magnitude than that of patients suffering from clinical disease. However, in very low endemicity areas, carriers will likely be symptomatic and the main source of transmission. The effectiveness of TBMV is determined by the degree of herd immunity induced in that part of the population responsible for transmission in a given area. The herd immunity induced by TBMV represents a variation from what is often thought of as herd immunity in than no members of the population are in fact protected against infection with the pathogen or the disease that it causes. Similar to all malaria vaccines the public health endpoint of a TBMV is reduction of the incidence of (severe) malaria. The biological endpoint is reduction or elimination of infected mosquitoes by blocking sporogonic development. Reliable assays are needed that are preferably close to the biological transmission-blocking effect or represent a good correlate. Pioneering work was conducted in the late eighties resulting in the development of the Standard Membrane Feeding Assay (SMFA), which is the gold standard for measurement of transmission-blocking activity in sera (282,283) (Fig. 3). In the case of P. falciparum, cultured gametocytes are used from a standard laboratory strain with stable gametocyte production (e.g., NF54). Because of the inability to culture P. vivax in the laboratory, packed cells from P. vivax–infected donors are used in the membrane feeding assay (284). The SMFA is believed to give a reasonable measure showing a fair correlation with natural feeding on gametocyte carriers (285). However, validation of the SMFA will depend on clinical trials showing acquired immunity. While an attractive assay because of its biological nature, interassay variation is clearly placing restrictions on the usage of the SMFA. However, when comparisons are made within experiments, the assay is appropriate for

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Figure 3 Standard Membrane Feeding Assay. Laboratory reared Anopheles mosquitoes are allowed to take a blood meal from membrane-covered glass devices that contain cultured gametocyteinfected red blood cell suspensions mixed with test or control serum (T or C). After feeding, the mosquitoes are maintained for six to eight days, and then are dissected to determine oocyst counts in mercurochrome-stained midguts. Transmission blockade is calculated by comparing oocyst numbers in a series of mosquitoes that are fed in the presence of test serum versus those fed with control serum.

detection of transmission-reducing activity in test sera (286). Another shortcoming of this time-consuming and laborious bioassay is the current limited capacity for large-scale testing of sera. Good correlations between antibody levels (as determined by ELISA) and transmission-reducing activity in the SMFA were shown for some of the major TBMV antigens (287; van de Kolk unpublished). This relationship will greatly aid in prescreening and down selection of TBMV during early development, since the ELISA is much easier to perform. Control of Malaria Transmission Malaria parasites spread in populations by Plasmodium-infected Anopheles mosquitoes. The intensity of malaria transmission is determined by the prevalence of gametocytes in endemic populations and the number of Anopheles mosquitoes in the area. Depending on the transmission pattern, malaria can be (i) present throughout the year in areas where transmission is perennial, (ii) seasonal following the onset of the rainy season and associated increase in mosquito breeding, and (iii) present as epidemics in areas where transmission occurs occasionally as a function of climatological fluctuations. Transmission intensity can be defined by either the Entomological Inoculation Rate (EIR) or Basic Reproduction number (Ro). EIR is the number of infectious bites per person/ year which can vary even within the same country in endemic areas of Africa from <1 to >1000 infectious bites/yr. Ro is the number of nonimmune individuals that can be infected from a single untreated and nonimmune malaria case and ranges from 1 to >3000 (288). Ro is directly related to vectorial capacity and malaria transmission is sustainable when Ro >1. Although the wet and warm parts of sub-Saharan Africa show perennial

intense transmission, sustainable levels of transmission may be unpredictable or generally absent in large parts of the continent that are dry or cool at altitudes >1600 to 1800 m where malaria transmission drops to Ro <10. In many parts of sub-Saharan Africa more than half of the population carries malaria parasites in the blood without becoming ill because of the acquisition of clinical immunity in childhood. These persons form an important reservoir for malaria transmission by mosquitoes. Large Anopheles mosquito populations often live near human habitats and regularly need blood meals for egg production. In fact, the principal malaria vector in Africa (Anopheles gambiae) only feeds on humans and is very efficient in transmitting malaria. The combination of abundant numbers of efficient mosquito vectors and a large reservoir of infected persons results in a high turn-over of parasites and intense infection pressure. Although the mechanism is not understood, there is a relationship between the incidence of (severe) malaria and transmission intensity. Obviously low transmission associates with low attack rates of disease but also a slow induction of clinical immunity and therefore a relatively high proportion of casualties. The objective of malaria control is to find a package of control measures that will reduce the risk for (severe) disease with minimal impact on NAI. Not every infection leads to disease and it is a challenge to separate both phenomena to allow for the built up of immunity. In the past there have been concerns that reduction of transmission per se would harm the acquisition of immunity and worsen or only shift the age of severe malaria (289). When transmission is very low, as is the case in most countries outside Africa, malaria can be quite effectively contained by residual household spraying with insecticides. In addition, with the introduction of long-lasting insecticide-impregnated bed nets and combination therapy, which includes artemisinin-based preparations that kill gametocytes and reduce transmission from men to mosquito, there is accumulating evidence showing that the incidence of clinical (severe) disease is significantly reduced over time (290–292). The long-term impact of these control measures and the impact in areas of intense malaria transmission is unpredictable since there are fewer examples of successful large-scale vector control programs in high-intensity areas. Coverage of artemisinin combination therapy and compliance may be lower in semiimmune individuals, because a substantial proportion of infections in these individuals are asymptomatic and any symptoms present are more likely to resolve with incomplete treatment than in nonimmunes (291). Thus it may be hard to achieve substantial reductions in transmission intensity, and the addition of an effective transmission-blocking vaccine could be of great benefit. Reduction of transmission intensity in a particular area has not been directly studied together with markers of malaria immunity in the population, so it is difficult to gauge the potential effect of a transmission-blocking vaccine on hindering the development of NAI. Serological markers have recently been shown to correlate with transmission intensity and may serve as an important tool to study changes in transmission intensity (293). For a rational epidemiological control strategy it remains important to determine the relationship between transmission reduction under a given intensity, presentation of clinical disease and effect on immune responses in longitudinal studies. There are clear indications, however, that transmission reduction possibly alone but more obviously as part of a larger control package, has a positive impact on malaria control.

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Interventions that specifically target malaria transmission will show differential effects on the presentation of clinical malaria depending on the transmission intensity (91). One can reasonably expect that such interventions will be become increasingly effective when the EIR is low (<8), preparing conditions for eradication. Targeting Hot Spots of Transmission with TBMV The population coverage that is needed for TBMV depends on the human gametocyte reservoir. The highest prevalence and densities are found in young children, which form an important transmission reservoir despite their relatively high prevalence of sexual stage–specific antibodies (294,295). Gametocytemia declines in the adult population because of lower presence of asexual parasites but frequencies are sufficiently high that this relatively large group also significantly contributes to transmission (296). Recent data show that detection of gametocytes by standard microscopy is grossly insufficient and that more sensitive molecular techniques indicate that the gametocyte reservoir is at least 3 to 30 times greater than previously thought (297–299). In principle, therefore, the entire population in a holoendemic area can drive transmission and qualifies as target for TBMV administration. The requirement of 100% population coverage has always been considered a major drawback for TBMV application. However, malaria transmission is determined by focal conditions and an accumulation of mini-transmission zones (300). Malaria distribution can vary substantially between villages and individual households (301–303). This variation reflects a composite of heterogeneities in vector distribution, human-vector contact and human host factors (304). The vicinity and productivity of mosquito breeding sites are key determinants of transmission intensity with mosquito dispersal generally being limited (305). In addition, household characteristics including structure, household crowding and personal protection measures against malaria play an important role in determining exposure to infectious mosquitoes (301,305). As a result, the relative risk of malaria is not uniform and can be over 10-fold increased in certain households (303). This clustering of malaria risk makes the certain households particularly affected by malaria, and consequently these households contribute disproportionately to malaria transmission. In other words, most mosquitoes acquire malaria in a limited number of houses that form ‘‘hot spots’’ of malaria transmission (Fig. 4).

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It has been estimated that typically 20% of the hosts are responsible for 80% of the disease transmission (303,306,307). Therefore 100% population coverage by TBMV may not be necessary to achieve protection at a community level if one can identify those households that are most important for initiating or maintaining malaria transmission (91). Control measures that target those who are most important for malaria transmission (i.e., who are bitten most) will disproportionately reduce transmission. Mathematical modeling suggests that with perfect targeting, herd immunity can be achieved by neutralizing a relatively small fraction of the human hosts, even at higher transmission intensities (287). Sufficient reduction of transmission may be achievable by targeting TBMV administration to population foci identified by satellites providing information on human habitat, vegetation and rainfall. TBMV, although sometimes criticized as an ‘‘altruistic’’ approach to malaria control, may not be as altruistic as perceived because administration of the vaccine to members of one focus of transmission (e.g., cluster of households) may directly result in reduction of disease incidence at the same microlevel. Thus MTBV can significantly contribute to personal protection, particularly when applied in low transmission areas where malaria transmission occurs on a microenvironmental level. Development of Transmission-Blocking Malaria Vaccines Specific antibodies against surface membrane proteins expressed during sporogony can effectively block transmission. Such antibodies bind shortly after ingestion of the blood meal to activated male and/or female gametes (prefertilization) or at a later stage after 20 to 24 hours to zygote/ookinete forms (postfertilization). Zygotes and motile ookinetes penetrate the peritrophic membrane and midgut epithelium to develop into oocysts on the basal lamina of the midgut outer cell wall. P48/ 45 and P230 are typical examples of prefertilization antigens, while P25 and P28 are typical postfertilization antigens, abundantly expressed on the membrane of zygotes and motile ookinetes. Prefertilization antigens are also expressed in gametocytes in the human host with the important possibility that boosting may occur upon natural infection. Postfertilization antigens lack this option since they are only expressed in the mosquito and vaccination needs to be such that long-lasting immunity is ensured without a requirement for natural boosting.

Figure 4 Targeting ‘‘hot spots’’ of malaria transmission to prevent transmission in a larger area. Houses with infectious mosquitoes are indicated in red and form the hot spots of malaria transmission (arrows). Control measures targeting these houses will have a dramatic effect through herd immunity.

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Antibodies can directly interfere with progression of sporogony, for example, with fertilization or migration through the midgut cell wall. Alternatively, parasites may be directly killed by complement fixed antibodies and/or ingested phagocytic cells (308,309). Since antibodies are responsible for transmission reduction, TBMV should be formulated to generate strong humoral immune responses with minimum variation in the range of elicited antibody titers. A narrow range of high antibody concentrations in the vaccinated population will significantly increase the efficacy of a TBMV (310). TBMV Candidates in Clinical Development Starting almost 30 years ago, the proof of concept of TBMV was demonstrated in animal models and in acquired sexual-stage immunity in human endemic populations that can prevent infection from man to mosquito (311–313). A number of proteins have been identified as potential targets and form the biological basis for the ongoing efforts to develop TBMV. For more than a decade the lead candidates have been unchanged and restricted to four proteins: Pfs48/45, Pfs230, P25 and P28 (where f = falciparum and s = sexual stage), the latter two being developed in both P. falciparum and P. vivax. Scientific, technological and financial hurdles are responsible for the slow progression of clinical development. The near absence of industrial interest and expertise in the field of TBMV has slowed down progression. A major technical stumbling block is the apparent and compelling need to produce properly folded proteins. All leading TBMV candidate proteins are relatively cysteine-rich with multiple disulfide bonds making B-cell epitopes conformation-dependent rather than linear. Only Pfs25 and Pvs25 have so far been tested in phase 1 clinical trials (314). Pfs48/45. Pfs48/45 belongs to a family of malariaspecific proteins that contain conserved motives with four or six cysteine residues. The protein is present on the surface of gametocytes, gametes, and zygotes (315,316). Pfs48/45 appears to resolve on SDS gels under nonreducing conditions in a protein doublet of 48 and 45 kDa, while under reducing conditions, only a single band of 58 kDa is observed. Alkylation of free cysteines results in one protein band (45 kDa), suggesting the 48- and 45-kDa bands are two different disulfide conformers. Five distinct B-cell epitopes with a subdivision for epitope II (IIa–IIc) have been defined on the basis of binding studies with a panel of Pfs48/45 specific monoclonal antibodies (317). Epitopes I–III in the C-terminal domain of the protein are conformational and epitope IV is linear. For epitope V in the Nterminal domain, both linear and conformation-dependent monoclonal antibodies have been described (317). Monoclonal antibodies to epitope I and V blocked transmission effectively in the membrane feeding assay but monoclonal antibodies to epitope IIa and epitope III were ineffective on their own but were able to reduce transmission when used in combination (318–321). An effect of serum complement on blocking activity of anti-Pfs48/45 antibodies has not been found. Mapping of the Pfs48/45 protein using antibodies, mutagenesis and limited proteolysis analyses revealed the presence of three domains: a N-terminal domain containing epitope V, a central domain comprising epitopes II and III, together with a C-terminal domain containing epitope I (322). The B-cell epitopes with the exception of epitope II appear not to be polymorphic, because amino acid substitutions

found in strains from various geographical regions do not affect recognition by the panel of monoclonal antibodies (321,323–325). A recent study reported 23 polymorphic residues in 44 P. falciparum strains of different geographic locations (326). A substantial numbers of them were found in one allele. Kenyan isolates have 12 polymorphic residues while Thai, Indian and Venezuelan isolates have 4, 8, and 9, respectively. There is an extreme geographical divergence of microsatellite allele frequency of Pfs48/45, specifically in position 253 and 254 representing the IIa and IIc variants as defined by monoclonal antibodies (324,325). Antibodies to both epitope IIa and IIc can be produced in natural infections. Epitope IIc is highly prevalent in Africa while IIa occurs in Asia and South American strains (325). Epitope I, IIb, II, and IV are conserved in Asian and African isolates (327,328). IIa and IIc variants have been identified in laboratory strains NF54 (IIc) and 7G8 (IIa), respectively. Variants otherwise do not affect binding of a panel of monoclonal antibodies that recognize five different epitopes (329). Whether the newly described polymorphisms affect epitope recognition is of interest and can be assessed when gametocyte-producing strains become available. Pfs48/45 is a target for natural immune responses as demonstrated by the presence of specific antibodies in field sera (318,328,329). There is no evidence that responsiveness against either Pfs48/45 or one of its epitopes is linked to particular HLA haplotypes but antibody prevalence in the population can be low (330,331). Data from Papua New Guinea show that seroconversion increases with age suggesting that immunological memory develops (328). Increasing anti-Pfs48/45 antibody titers are also observed after long exposure to gametocytes in Dutch expatriates (Roeffen, unpublished) and after repeated malaria infection following migration of malaria-naive individuals into a hyperendemic region (332). Antibody titers for the individual epitopes show considerable differences between and within field samples (328). Although a reasonable assumption, it is unknown so far whether anti-Pfs48/45 antibodies in natural sera contribute to transmission-blocking activity. A significant correlation has been shown between the presence of anti-Pfs48/45 antibodies and transmission-blocking activity in some studies, although this finding has been absent or less convincing in others (328,330,333). Differences in methodology for antibody detection and in malaria endemicity may be responsible for this nonconcordance. The limited persistence of Pfs48/45 in macrogametes/ zygotes and complete block of ookinete formation by antiPfs48/45 monoclonal antibodies indicate that transmission is blocked at the level of fertilization and zygote formation (319). Although present in male and female gametocytes and macrogametes, the location of Pfs48/45 on the surface of P. falciparum microgametes is not firmly established. Pfs48/45 plays a pivotal role in zygote formation (334). Disruption of the P48/45 gene showed that gametocytogenesis and gametogenesis proceeded normally but development of the mosquito stages was strongly reduced, though not completely absent. It was shown that Pbs48/45 male but not female gametes were incapacitated for zygote formation (the orthologous protein in the rodent parasite P. berghei). Thus Pfs48/45 is an attractive vaccine candidate but the conformational nature of the transmission-blocking B-cell epitopes, the abundant presence of cysteines and the multiple potential glycosylation sites have been major stumbling blocks

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for production of sufficient quantities of properly folded recombinant protein in a variety of pro- and eukaryotic expression systems (322,335). However, simultaneous coexpression with four periplasmic folding catalysts in E. coli resulted in expression of properly folded C-terminal Pfs48/45 fragment fused to maltose binding protein. This fragment that contains epitope 1 induced consistently high titers of transmissionblocking antibodies in mice and provides a solid basis for clinical development of a Pfs48/45 TBMV (335). Pfs230. Similar to Pfs48/45, Pfs230 is a member of the family of malaria-specific proteins with a 6 cysteine motif containing 16 tandem copies of this motif (336). Pfs363-kD is a precursor protein that is processed to a 300- or a 307-kD molecule when gametocytes are activated. These fragments do not contain GPI membrane anchor moieties but partly form a membrane complex with Pfs48/45 (337–339). The cleaved amino terminal peptides are released in the environment (337,340). Pfs48/45 may be essential for the membrane localization or processing of Pfs230 since Pfs48/45 gene knockout parasites produce Pf363 but do not express Pfs230 on their surface (334). In contrast, Pfs48/45 (as well as Pfs25) is normally expressed in Pfs230 gene knockout parasites, although oocyst production is significantly reduced (not blocked) probably because of diminished formation of exflagellation centers (constituted of male gametes and red blood cells) preceding fertilization of macrogametes (341). Parasites with disrupted Pfs230 are resistant to the alternative complement cascade. The exact function of Pfs230 remains elusive but clearly relates to this male gamete/red blood cell interaction. Five different B-cell epitopes have been identified spanning the entire protein. Irrespective of the epitope localization, monoclonal antibodies made to each of the five epitopes can block transmission under the condition that the isotype is complement-fixing and that active complement is present (339,342–344). In vitro studies show that P. falciparum macrogametes undergo lysis suggesting that mere binding of complement fixed antibodies may explain the mechanism of Pfs230mediated transmission blockade; however, monoclonal antibodies against Pgs230 of P. gallinaceum block transmission in the absence of complement suggesting that alternative mechanisms may be in place (345). Pfs230 expressed by gametocytes is immunogenic under field conditions and specific antibodies are found in a large proportion of endemic sera with differential recognition of Pfs230 fragments (329,339,346–349). Since Pfs230 like Pfs48/45 is only expressed intracellularly in gametocyte-infected red blood cells, these antibodies only can bind to gametes in the mosquito midgut. There is evidence that such antibodies can reduce sporogony, but the association with transmission blockade remains controversial most likely because of methodological differences (329,339,346–349). The only report of a recombinant Pfs230 protein fragment inducing some transmission-reducing activity dates back more than 10 years ago. In this case, most likely only a minor fraction of the Pfs230 protein fragment was properly folded because of the large number of cysteines. Several other expression systems showed the same shortcoming and were unable to produce the desired natural conformations (350). A solution may be found in the coexpression of chaperones, as was successfully shown for Pfs48/45 (335). P25 and P28. P25 and P28 are structurally related postfertilization antigens with exclusive protein expression on

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the surface of zygotes and ookinetes. P28 is probably expressed later than P25 in sporogonic development (351). The genes for both proteins have been successfully isolated in both P. falciparum and P. vivax (352,353). Both antigens contain four epidermal growth factor (EGF)-like domains and a GPI anchor sequence. The function of the proteins has not been clarified but there is evidence that they may protect the parasite against the mosquito defense system (354). Monoclonal and polyclonal antibodies directed against either P25 or P28 effectively block infection of mosquitoes (281,284,319,355). A linear epitope of Pfs25 was identified by using transmission-blocking monoclonal antibodies (356). Combination of P25- and P28specific antibodies may result in an additive effect on transmission blockade (357). Anti-P25 and anti-P28 antibody activity is complement independent for P. falciparum but possibly not in the case of P. vivax (358). Specific antibodies to these proteins may damage the parasites, but most likely, their effect is the prevention of ookinete migration through the midgut (359–361). Disruption of P25 and P28 genes in murine malaria results in significant reductions but not complete blockade of oocyst development (362). Only when both genes are deleted is complete arrest obtained, suggesting a mutual functional complementarity. Still antibodies to the individual proteins can completely block transmission but relatively high concentrations are needed. These data strengthen the argument for a postfertilization TBMV consisting of both P25 and P28. The absence of anti-Pfs25 antibodies in human sera from malaria endemic areas and the limited genetic polymorphisms and minimal variation in its amino acid sequence, suggest that the relative homogeneity is due to the absence of immune pressure (353,362–365). While the former seems to exclude the possibility of boosting by natural infections and thus could be perceived as a disadvantage, limited variation accommodates a major issue for many malaria vaccine target proteins. Pfs25 in P. falciparum and Pvs25 in P. vivax are the clinically most advanced TBMV. Clinical-grade Pfs28 has only been produced as a fusion protein with Pfs25. Surprisingly, immunogenicity studies in rabbits showed that this fusion protein was less potent in eliciting transmission-blocking antibodies than the single Pfs25 recombinant protein (366). Target specific immunogenicity was disappointing when E. coli–expressed P25 proteins were used but improved in eukaryotic expression systems (367,368). Clinical-grade material of Pvs25 and Pfs25 has been produced in yeast eliciting transmission-blocking antibodies in mice, rabbits and nonhuman primates. Both Pfs25 (TBV25H) and Pvs25 (Pvs25H) have been tested in dose-finding phase 1 clinical trials (358). The first human TBMV trial was conducted in 1994 with a TBV25H formulation adjuvanted with alhydrogel (369). A hypersensitivity reaction occurred in one volunteer probably because of formulation conditions and inadequate absorption of the protein to aluminum. Later, Pfs25 was incorporated into a recombinant vaccinia virus containing several malaria proteins (NYVAC-Pf7). Albeit not sufficiently potent, postvaccination sera showed presence of transmission-reducing activity in the SMFA (369). The poor immunogenicity of TBV25H was improved by changing the Pfs25/alum formulation. Co-administration of CpG oligodeoxynucleotides or conjugation to secondary carriers induced long-lasting and significantly higher titers of anti-Pfs25 antibodies in experimental animals (370–372). Using Pfs25 DNA plasmids it was shown that the technique

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of immunization also can have a profound impact on the elicited immune response; up to a hundred fold less Pfs25 plasmid DNA was needed for comparable antibody titers in mice by using electroporation rather than intramuscular injections (373). More than 10 years after Pfs25, the first phase 1a trial with Pvs25H was conducted in thirty healthy volunteers. Pvs25 adsorbed to alum was well tolerated, generating transmission, reducing antibodies, which correlated with ELISA titers (358), a correlation which has been confirmed by more recent work (287). A second trial assessed Pfs25 and Pvs25 formulated, individually, with Montanide ISA 51. The trial was halted because of systemic reactogenicity including erythema nodosum and leukemoid reaction, although Pfs25/ISA 51 induced transmission-blocking antibody responses (374). New formulations are now under development at the NIH in the United States. Other efforts to develop transmission-blocking vaccines are being pursued at the Johns Hopkins School of Public Health in Maryland (373), Nijmegen Medical Center, The Netherlands (335), and the Ehime University in Matsuyama, Japan (375). From the combined data, it is clear that P25 and P28 immunogenicity needs to be improved to ensure that sufficient quantities of functional antibodies are generated. The recent options illustrated by data with Pfs25 in animals may offer a solution not only for postfertilization antigens but also for prefertilization proteins. Particularly encouraging for the latter is that vigorous boosting occurred by the unconjugated protein following priming with the protein-carrier conjugate suggesting the possibility of natural boosting (371). Concluding Remarks Priority in malaria vaccine development has been primarily given to pre-erythrocytic stages and asexual stages because they directly target the emergence of parasitemia and disease. In contrast, investments in the development of TBMV have lagged behind because gametocytes do not induce morbidity and mortality. In the coming years, however, TBMV may gain an increased priority not the least because of a recent call for a new campaign to eradicate malaria (376). TBMVs alone or as part of a multistage vaccine or other control strategy will likely be an effective and important component of such eradication efforts (91). TBMV development has focused on P. falciparum because this species is primarily responsible for the high disease burden in sub-Saharan Africa. In virtually all other malaria areas in the world, however, both P. falciparum and P. vivax are present together. Since P vivax requires less stringent conditions for transmission than P. falciparum, a successful campaign against P. falciparum may result in an unwanted increase in P. vivax burden (377). Thus, TBMV application can only be envisaged in such areas if both species are covered (378). To date, only a small handful of vaccine candidate proteins has entered clinical development. Following the completion of the genome sequencing projects for P falciparum and more recently P vivax, additional sexual-stage proteins will be identified as potential TBMV targets (314,379). An additional notable development is the finding that antibodies to mosquito midgut proteins may also reduce transmission by membrane feeding assay (380,381). In the coming years the primary challenge for TBMV will be formulation to enhance immunogenicity (382). Design and execution of TBMV field trials is an unprecedented area with a need for careful exploration.

MALARIA VACCINES: THE TECHNOLOGY ROAD MAP In October, 2004, a team of experts assembled to focus the energy and vision of the malaria vaccine community by defining a roadmap for vaccine development. This effort, eventually involving 230 scientists and health professionals from 35 nations, was sponsored by the Bill & Melinda Gates Foundation, the PATH Malaria Vaccine Initiative, and the Wellcome Trust, with additional direction provided by the World Health Organization. Various groups met in a series of meetings held over the course of nine months, culminating in the publication of a report, the Malaria Vaccine Technology Roadmap, in August 2006 (383). Among other recommendations, this report supported the importance of basic research, including the identification of new antigens, particularly those acting at the interface between host and parasite, and the characterization of their biological function. The rationale was that understanding biological function should reveal strategies for intervention, and there are many supporting examples indicating that this is the case (384,385). Although this approach is clearly appropriate, much of what we know about parasite biology indicates that key pathways may be redundant; whether it is homing to the liver or invading erythrocytes, there appear to be multiple molecular interactions occurring in sequence or in parallel that allowing the parasite to thrive. For example, blocking erythrocyte invasion by P. falciparum has revealed a second pathway that is nearly as efficient for mediating entry (386,387). Thus, not just a thorough understanding of biology at the molecular level, but also the availability of vaccine technologies allowing the combination of multiple antigens to block multiple pathways, may be required to realize the potential of this approach. Additional Roadmap recommendations included standardizing immunological assays and clinical trial designs. The architects of the Roadmap recognized that this would be required to facilitate the comparison and down selection of vaccine candidates. However, as a measure of the uncertainty in the field regarding the pathway to an effective malaria vaccine, no specific recommendations could be made regarding platforms or approaches. It was suggested that a systematic algorithm be established for prioritizing subunit vaccine candidates using accepted preclinical criteria, but until there are several vaccines identified that offer substantive protection in the field, forming a link between preclinical findings and protection, it is difficult to know what those criteria might be. We have no established in vitro measures of protective immunity outside of the transmission-blocking assay, and no satisfactory animal models where P. falciparum vaccines can be assessed for protection. On the basis of these considerations, the specific development recommendations coming from the Roadmap were restricted to a statement that a diversity of approaches should be tested, including multistage, multiantigen subunit vaccines as well as whole organism vaccines.

Strategic Approaches It is thus clear that the field of malaria vaccine development represents a scientific frontier. In the absence of clear answers regarding the approach to take, there is a need for champions to pursue particular approaches to their logical conclusion, to see if they work. Since there are no vaccines licensed for the prevention of chronic parasitic infections, and very few

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impacting chronic viral or bacterial infections, we have no successful examples to follow. Which Antigens, and How Many? The most advanced vaccine currently in development, RTS,S, illustrates the dilemma posed by the ‘‘antigen question.’’ Even as the vaccine enters phase 3 licensure trials (388), strategies are being devised to improve its efficacy through combination with other proteins (113), new adjuvant formulations (389), or heterologous prime-boost regimens with other vaccine platforms (154,390–392). However, it has proven difficult to achieve substantive improvements. Does RTS,S represent the limit of what can be achieved with a single antigen? Or can breakthroughs still be achieved and the protective efficacy boosted to significantly higher levels? Further along the antigen spectrum is the multiantigen subunit approach, represented first by the vaccinia construct NYVAC-Pf7 (393,394) and subsequently by Combination B (255), MuStDO 5 (395), ME-TRAP (162), L3SEPTL (396), PEV3A (397) and several other vaccines previously or currently under development, in which the combination of multiple antigens or epitopes from multiple antigens is intrinsic to the design of the vaccine. Multicomponent subunit vaccines can be complex to formulate and costly to manufacture. Antigenic components may interfere with each other, reducing the potential for synergy (398). However, both models of protective immunity—IS and NAI—indicate that multiple antigens may be required to achieve high-level protection (399). Combinations may provide synergistic interactions that are not evident if the individual components are examined individually. In sum, does this represent a more profitable approach than working to perfect a single protective antigen? At end of the antigen spectrum lie the whole organism vaccines. Two methods of sporozoite attenuation—diffuse genetic damage by radiation, or focal knockout of discrete genes required for development—generate an immunogen that in mouse, nonhuman primate or human models affords >90% protection against sporozoite challenge (95,400–403). The potentially hundreds of antigens responsible for the protection are not defined, but if an attenuated sporozoite or blood-stage vaccine is developed for practical administration and provides solid protection, does knowing the antigen composition even matter? This empirical approach stands in contrast to the building-block approach favored by subunit vaccine developers as they elucidate critical biological processes underlying parasite development and attempt to disrupt them by combining key antigens into a single vaccine. Equally important to the number of antigens to be included in the vaccine is deciding which part of the antigens to include. Given the importance of T-cell responses to protection, and their genetically restricted nature, many believe that it is important to include full-length antigens as a way to maximize the number of epitopes available to an MHCdiverse human population. On the other hand, there is evidence that intact proteins may render protective epitopes cryptic to the immune system (404), and this would argue that the regions of antigens selected for inclusion in a vaccine have to be carefully selected as being optimal on the basis of experimental evidence. An additional dimension to antigen question is represented by the need for antigen discovery. Traditional vaccine antigens represent <0.2% of the genome, and there is a growing consensus supporting the systematic identification

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and testing of novel antigens. Several programs are currently engaged in this effort. There remains the attractive possibility that current vaccine formulations are adequate for affording high-level protection, but we just need to identify novel highly protective antigens to replace the current candidates. On the other hand, our current antigens may be adequate, and the main problem may be that vaccine delivery systems are insufficiently potent. This is precisely the question faced by the developers of blood-stage vaccines posed earlier. Which Vaccine Platform or Platforms? This also introduces the second major question, which is: what is the optimal delivery platform for the selected antigens? As described, the diverse biologies underlying the parasite life cycle imply that multiple immunological mechanisms may contribute to protection against various life cycle stages. Recombinant proteins are particularly effective at inducing T-cell help and antibody production, while genetic approaches, in particular viral vectors (405) and DNA vaccines (406,407), are more potent for expanding CD8þ T-cell populations and associated CTLs (see chap. 70). At this point, for all the reasons provided, we do not know which immunological mechanisms will ultimately enable the induction of solid and long lasting protection. Thus a consideration of all the various vaccine platforms is currently indicated.

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70 Gene-Based Malaria Vaccines Adrian V. S. Hill The Jenner Institute, University of Oxford, Oxford, U.K.

INTRODUCTION

PRECLINICAL STUDIES

The development of gene-based malaria vaccines represents one of three major current approaches to malaria vaccine development. The largest effort is currently on the design and development of protein/adjuvant vaccines, as illustrated by the RTS,S/AS01 candidate (1). This is also the most popular approach for the development of blood-stage vaccines for which antibody-mediated immunity is widely regarded as the major protective mechanism. Gene-based vaccines have been in clinical trials for malaria since the assessment of NYVAC-Pf7 in the mid-1990s (2), and most recently, a major effort has been initiated on a third quite different approach, the development of whole-parasite vaccines (3), but the clinical assessment of such vaccines using deployable routes of administration has yet to begin. Moreover, there are concerns that even if successful, whole-parasite vaccines will likely require transportation and storage in liquid nitrogen vapor, limiting their deployability. In their short history, gene-based malaria vaccines have made some notable contributions that are of relevance to vaccinology as a whole. The first published report of the safe human use and T-cell immunogenicity of plasmid DNA vaccines was in the malaria field (4). Shortly afterward, the first use of two gene-based vaccines in a clinical trial was undertaken for malaria, demonstrating in humans the potential of heterologous prime-boost immunization for high-level T-cell induction (5). Arguably, the only clear demonstration that T cell– mediated protection from infection can be induced in humans in the absence of antibodies has emerged from the same vaccination approach. And there is now increasing evidence for a variety of malaria vaccine trials that cellular immune correlates of protection can be identified (6,7), encouraging further development of this approach. Although the headline efficacy of gene-based malaria vaccines has yet to match that of the sole partially successful protein-adjuvant candidate, the remarkable flexibility and rapidly improving clinical immunogenicity data with the latest gene-based vaccines suggest that this technology probably has the greatest chance of producing a highly effective, widely deployable malaria vaccine in the medium term. This prospect has been enhanced by the outstanding safety record of genebased vaccines in clinical trials to date and by substantive advances in the scalability of manufacture of several viral vector-based platforms. I shall review first the preclinical data on a variety of gene-based vaccines before summarizing the rapidly increasing body of clinical trial data in this field, and end with some thoughts on prospects for the development of this field.

Although attempts to use whole-parasite and parasite extracts as malaria vaccines date back to at least the 1940s (8), and clinical trials of protein-based subunit approaches to the mid1980s (9), the first DNA-based approach was reported using the Plasmodium knowlesi circumsporozoite (CS) protein in 1984 (10). A recombinant vaccinia virus expressing this antigen was used to induce antibodies in mice and rabbits that recognized sporozoites. Notably, this work was undertaken at a time of great optimism about the feasibility of a malaria vaccine being available imminently, following the cloning of many malaria antigens in the early to mid-1980s. It was recognized early that recombinant viruses might have an advantage over peptideconjugate vaccines, seen then as the leading competing technology, through their ability to induce strong T-cell responses. This was supported by the demonstration in 1988 that cytotoxic T lymphocytes to the Plasmodium falciparum CS protein could be induced by a recombinant vaccinia virus (11), and the relevance of T-cell immunity to the strong protection induced by irradiated sporozoite immunization in mice was highlighted by several studies (12,13). A further potential application of recombinant vaccinia vectors in malaria was illustrated by Kaslow et al. (14), who demonstrated the induction of transmission-blocking antibodies against a sexual-stage antigen, Pfs25, in mice. However, less encouraging efficacy data were reported in immunization studies of squirrel monkeys, which are susceptible to P. falciparum. Recombinant vaccinia vectors expressing some or all of four blood-stage antigens, MSP1, MSP2, AMA1, and RESA induced only modest antibody responses and no protection against parasite challenge (15). Several groups attempted unsuccessfully to induce protection against sporozoite challenge using recombinant vaccinia expressing the CS protein from the well studied rodent malaria parasites Plasmodium yoelii and Plasmodium berghei (16,17) but, surprisingly, high-level protection was found using a NYVAC strain (18). This latter work, which was part of a substantial collaboration between the Walter Reed Army Institute of Research and the Virogenetics biotech company, led to the generation and assessment of several recombinant malaria poxvirus constructs based on the NYVAC and ALVAC backbones. NYVAC is a derivative of the Copenhagen strain of vaccinia rendered replication-defective through deletion of 18 open reading frames by molecular engineering (19); ALVAC is strain of canarypox virus and therefore also replication incompetent in mammalian cells. An impressive seven-antigen construct was then developed in NYVAC. This included the genes for the preerythrocytic antigens CS and thrombospondin-related adhesion

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protein (TRAP) and LSA1, the blood-stage antigens MSP1, AMA1, and SERA, and the sexual-stage antigen Pfs25 (2), which was progressed to a phase I/IIa clinical trial (see below). Modified vaccinia Ankara (MVA) recombinants were developed for malaria by the Oxford University group because this nonreplicating vector had been safely used in more than 100,000 persons as a smallpox vaccine, was free of the intellectual property constraints of the NYVAC and ALVAC vectors, and had potential for large-scale low cost manufacture on chicken embryo fibroblasts or new cell lines (20). Surprisingly, MVA was found to be more immunogenic for T-cell induction than replicating vaccinia strains (21), but in small animal models, MVA used alone still induced minimal protection unless heterologous prime-boost regimens were employed (see below).

DNA Vectors The first use of plasmid DNA as vaccines against the preerythrocytic stages of malaria was reported by the U.S. Navy group (22,23). Both T cells and antibody responses were induced to the CS protein of P. yoelii, and a protection rate of 68% was reported against sporozoite challenge (23). This result generated great interest in the potential of DNA vectors as vaccines against malaria. P. falciparum constructs were generated, and extensive safety studies undertaken by the Navy/Vical collaboration aimed at clinical testing of DNA vaccines. Further studies suggested that these early estimates of efficacy in mice might have been too high, with later studies by the same group reporting protection rates of less than 25%. This led to several attempts at improving the efficacy of DNA vaccines, not only with the use of a viral vector boost (see below) but also the assessment of plasmid-encoded cytokine adjuvants such as granulocyte-macrophage colony-stimulating factor (GM-CSF), which appeared to enhance immunogenicity and efficacy at least at low plasmid doses (24). However, the general experience with assessment of potential DNA vaccine adjuvants in malaria models has been that few if any of a very large number of potential adjuvants tested have induced consistent enhancements of immunogenicity. It was also noted that protection differed between mouse strains, at least in part attributable to different major histocompatibility complex (MHC)-restricted epitopes. This was addressed in part by adding further antigens to plasmid DNA mixtures (25). After initial evidence that this could enhance efficacy, along with some evidence of protection against blood stages with plasmid DNA, ambitious plans were generated to assess five antigen and also 15 antigen mixtures in clinical trials. This has involved extensive testing of potential interference by particular malaria antigens in mixtures, and clear evidence of adverse effects of particular antigens on the immunogenicity of others was provided (26). Other groups had less success in demonstrating any protection using DNA vaccines to protect against the preerythrocytic stages of P. berghei using either intramuscular DNA injection (21) or intraepidermal delivery using a ballistic device (a ‘‘gene gun’’) (27). Despite the lack of efficacy of DNA vaccines in clinical trials for malaria, there is still interest in using this as an approach to identify potentially protective antigens in large mixtures. Such ‘‘expression library immunization’’ (28) has sought to exploit the availability of large numbers of antigens defined through whole genome sequencing efforts to identify antigens more protective than those widely studied in current efforts.

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Heterologous Prime-Boost Approaches An increasingly widely used approach to inducing stronger Tcell responses with vectored vaccines is to use sequential immunizations with two vectors each expressing the same antigen. This prime-boost strategy dates back to the discovery of enhanced efficacy against P. yoelii sporozoite challenge by priming with a recombinant influenza virus and boosting with replication–competent vaccinia virus (29). The order of the immunization was found to be important for protection. Although T-cell responses were not measured after immunization, the protection induced was shown to be dependent on CD8 T cells. The same enhanced protection was found by Schneider et al. using nonreplicating vectors, plasmid DNA and MVA, in P. berghei studies (21). Again, the order was critical with DNA effective as a priming agent but not as a booster. Schneider et al. (21) used ELISPOT as well as lysis assays to show that the protection was dependent on reaching a threshold level of T-cell immunogenicity, a result also shown using recombinant Ty virus-like particles to prime and MVA to boost (30). The DNA-poxvirus strategy was extended to the P. yoelii model with the use of an alternative nonreplicating poxvirus, NYVAC (or COPAK), to enhance immunogenicity and efficacy (31). Cockburn et al. (32) have recently analyzed the mechanisms whereby vaccinia vectors provide a more potent boost than adenovirus (Ad) or influenza vectors. and show a requirement for dendritic cells and an ability of vaccinia to overcome feedback inhibition by CD8 T cells to allow better antigen presentation, an interpretation consistent with in vivo imaging studies of antigen expression (33). These murine studies were then extended to nonhuman primates. In chimpanzees, boosting of a DNA-primed response with a recombinant MVA induced higher T cell responses, that appeared to be CD4þ, than use of DNA alone (34). Rogers et al. generated DNA and poxvirus vectors based on the ALVAC canarypox strain encoding two pre-erythrocytic (CSP and TRAP/SSP2) and two blood-stage antigens (MSP1 and AMA1) from the monkey parasite P. knowlesi (35). Enhanced antibodies and T cells were observed after the poxvirus boost, and some limited protection against sporozoite challenge observed, apparently mainly focused on the pre-erythrocytic stages. A further similar study but using the NYVAC/COPK vaccinia vector to boost showed somewhat greater protection, but again, with a clearer pre-erythrocytic than blood-stage effect (36). In both studies, antibody responses as well as T-cell responses were substantially enhanced by the poxvirus boost. A recent more extensive study by Weiss et al. (37) in rhesus macaques provided clear evidence that the blood-stage antigen (MSP1 and AMA1) expressing vectors were contributing to protection, but the precise immunological mechanism remains to be defined. There was some evidence that a longer interval between DNA priming and the poxvirus boost could enhance efficacy in these macaques. In mice, this was seen in the P. yoelii model (38) but not evident in studies of DNA-MVA immunization against P. berghei (A. Moore and A.V.S. Hill, unpublished data). Little has been reported on the use of DNA-poxvirus or avipox-orthopox (such as fowlpox-MVA) prime-boost regimens in murine models of blood-stage malaria, despite this evidence (37) of potential efficacy in nonhuman primates. Draper, Biswas, Hill, et al. (unpublished) studied DNA-MVA regimens in P. yoelii in mice and failed to generate protection even though adenovirus-MVA regimens were strongly protective (see below). Evidence that multistage protection can be achieved in nonhuman primates with vectored vaccines has increased

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interest in using combinations of antigens. Original plans to use up to 15 DNA vaccines (in the U.S. Navy Must Do program) have been simplified to a five-antigen (CSP, TRAP, LSA1, MSP1, and AMA1) approach using both DNA plasmids and a canarypox vector encoding these five plus two other antigens. Jiang et al. found no evidence of interference between the five antigens when used as a mixture in macaques (39), although significant inhibition has been found in earlier murine studies of a nine-plasmid mixture (26). These studies confirmed that the majority of T cells induced with DNA-poxvirus in macaques are CD4þ rather than CD8þ, and more contained IL2 on flow cytometry than interferon-g. The limited efficacy of DNA-MVA regimens in early clinical trials led to attempts to improve on this regimen by substituting another viral vector for the DNA component. Anderson et al. studied an FP9 strain of fowlpox, which appears to consistently induce better CD8 responses than the standard Webster’s strain (40,41). In the P. berghei murine model, FP9MVA regimens induced better CD8 T-cell immunogenicity and better efficacy than DNA-MVA regimens. However, few nonhuman primate data are available on such avipox-orthopox primeboost regimens in malaria. These impressive immunogenicity and efficacy results in malaria animal models led to early clinical studies of heterologous prime-boost regimens in malaria (5), and subsequently in HIV, tuberculosis, and other disease areas.

Adenovirus Vectors In 1997, the use of adenoviruses to induce protective immunity against P. yoelii malaria was reported (42). Levels of CD8 T cells higher than achieved with any other vector could be induced to the CSP with a single immunization of a recombinant based on the standard serotype five vector, leading to substantial protection against sporozoite challenge. Higher numbers of antigenspecific T cells were induced in the liver than the spleen by this vaccine, which had been rendered replication incompetent in mammalian cells by deletion of the E1 gene. This result was comparable to the efficacy of a single-dose of irradiated sporozoites delivered intravenously (43); interestingly the Adinduced protection was not dependent on interferon-g (44). In the P. berghei model, adenovirus was found to be capable of both priming and boosting protective CD8 T cells against the CSP (45). In heterologous prime-boost regimens, adenovirus was a more immunogenic and protective vector than plasmid DNA or Ty virus-like particles, and of the many regimens compared, adenovirus priming and MVA boosting was the most protective (45). In P. yoelii, adenovirus prime-vaccinia boost regimens were found to be more protective than the adenoviral vector alone, and led to more durable immunity (46). Studies of the effects of deleting the glycosylphosphatidylinositol (GPI) anchor signal sequence from the CS protein sequence of P. yoelii and P. falciparum both indicated that this could lead to enhanced antibody and T-cell immunogenicity, likely related to altered cellular processing of the truncated antigen (47,48). Because of the high prevalence of antibodies to the Ad5 serotype in Africa, attempts have been made to use alternative serotype vectors. Crucell has explored the use of an Ad35-based vector, based on a prevalence of anti-vector antibodies of 20% to this strain in Africa compared with 80% for Ad5 (49,50). Although an Ad35 vector expressing P. yoelii CSP was less potent than the corresponding Ad5 vector, it was more protective in the

presence of antibodies to the latter serotype. Also, a prime-boost regimen of Ad5-Ad35 and Ad35-Ad5 vectors was more potent and protective than single vector regimens (50). Most rare human serotypes that have been assessed as adenoviral vectors appear substantially less potent than Ad5. However, chimpanzee vectors have been identified that are at least as potent as Ad5 (51). In mice single dose protection could be obtained against P. berghei using the C6 simian vectors encoding CSP (52). Use of a full-length CMV promoter with intron A was found to be important to maximize immunogenicity. Also, use of the multiple epitope-thrombospondin-related adhesion protein (METRAP) insert, which retains only a single nonamer CD8 epitope from P. berghei yielded excellent protection in a variety of chimpanzee vectors (51). Higher level and better-sustained protection could be induced by simian adenovirus prime MVA boost regimens, which also enhanced the frequencies of polyfunctional CD8 T cells (53). Prevalences of neutralizing antibodies to chimpanzee serotypes in Africa appear lower than to Ad35 suggesting that this should not be a significant hindrance to the use of these vectors for malaria (54). Recently the use of adenoviruses as blood-stage vaccine vectors has been investigated by Draper et al. who found that adenovirus-MVA regimens expressing MSP142 could induce high-level protection in mice against P. yoelii in mice. Although strong T-cell responses were induced, blood-stage protection was mediated by high-titer antibodies (55). Vaccine efficacy was higher against sporozoite challenge related to partial T cell–mediated protection at the liver-stage (56). In studies of P. falciparum antigens high-titer antibodies with substantial growth inhibitory activity could be induced with both MSP1 and AMA1 inserts suggesting that a multistage vaccine based on this adenovirus-MVA regimen should be feasible (Draper, Biswas, Hill, et al. unpublished).

CLINICAL STUDIES DNA Vectors The first report of a clinical trial of DNA vaccines in humans was of the CSP gene undertaken by the U.S. Navy group (4). Although no antibody responses were observed (57) cells from many vaccinees could be re-stimulated in vitro to generate HLA class I–restricted cytotoxic T cells. Up to about 100 spot forming cells per million PBMCs could be detected to some peptides in ELISPOT assays and responses were somewhat higher when the Biojector jet device was used rather than a needle and syringe (58). A study of the TRAP sporozoite antigen linked to a string of mainly CD8 T-cell epitopes from multiple malaria antigens also showed modest immunogenicity when administered by needle intramuscularly. The Oxford group compared this route to administration of low microgram amounts of DNA on goldbead administered to the skin and observed similar immunogenicities (5). Some volunteers, immunized with the ME-TRAP plasmid DNA, were challenged with five mosquito bites and no evidence of protection was observed (5). The U.S. Navy group proceeded to assess a mixture of five plasmid DNAs encoding the pre-erythrocytic CSP, TRAP/ SSP2, LSA1, LSA3 and Exp1 genes (59). Some individuals also received a plasmid expressing GM-CSF in an attempt to enhance immunogenicity. However, again T-cell responses were modest and actually lower in those receiving GM-CSF. No protection was observed against sporozoite challenge although this was observed to boost DNA-primed immune responses.

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In summary both single antigens and mixtures of plasmids showed moderate T-cell immunogenicity, minimal antibody responses and no evidence of protective efficacy in humans.

DNA Vectors in Prime-Boost Regimens On the basis of the enhanced immunogenicity and efficacy of DNA-MVA regimens in mice the ME-TRAP construct was assessed in a dose escalating prime-boost regimen using intradermal MVA administration, a route suggested by preclinical (60) comparisons. In all cases, heterologous prime-boost regimens showed greater T-cell immunogenicity than single vectors (5), a principle now exemplified in several other diseases (61–63). Higher doses of MVA were more immunogenic. Delivery of DNA was compared using intramuscular needle and syringe with a ballistic ‘‘gene gun’’ device delivering a microgram of DNA on gold beads. The immunogenicity of these approaches appeared similar despite the ballistic device delivering much smaller quantities of DNA. A limited comparison of prime-boost intervals suggested that a three-week interval between the last DNA immunization and the MVA boost was as effective as an eight-week interval. Administration of a second MVA dose close to the first did not enhance immunogenicity, but a field study later showed good boosting with an interval of a year between MVA administrations (64). Boosting of a DNA-primed immune response enhanced Tcell immunogenicity from about 50 to 100 SFU/million to in one case a mean of over 1000 SFU/million (5). Most of these responding T cells were CD4þ and as many contained IL-2 as interferon-g (65). In several challenges there was an overall delay in time to parasitemia and in a DDM regimen one out of eight volunteers showed sterile protection. This individual had the highest T-cell response of that immunization group and overall regimens that induced stronger responses were more protective. Importantly, antibody responses induced were either very weak or absent implicating cellular immunity in protection, arguably the first demonstration that any subunit vaccine can protect humans in the absence of antibody induction. A collaborative study between the Oxford and U.S. Navy groups allowed a head to head comparison of the immunogenicities and efficacy of DNA-MVA regimens encoding the METRAP and CSP inserts (66). The CSP constructs were found to be less immunogenic than those encoding ME-TRAP and only the latter showed some protection on sporozoite challenge. It remains unclear why CSP performed more poorly than METRAP. Possibilities include a lesser immunogenicity of the CSP sequence for T-cell induction, perhaps reflecting selection pressures, or some design feature of the CSP construct, including the possibility that inclusion of a GPI anchor sequence at the Cterminus was deleterious to immunogenicity (47,48). The DNA-MVA regimen was assessed in phase I trials in adults and young children in The Gambia and, on the basis of excellent safety and good immunogenicity data, progressed to a phase IIB trial in Gambian adults in late 2002. Analyzing time to disease in a cohort of Gambians followed through a malaria season only a nonsignificant efficacy of 11% was observed (67). Estimation of the degree to which this DNA-MVA regimen could reduce liver parasite burdens in sporozoite challenged vaccinees indicated about an 80% reduction in parasite load (5,68). Taken together with the Gambian phase IIb trial result this suggests that pre-erythrocytic vaccines will have to perform at a higher level in sporozoite challenge studies to attain useful field efficacy.

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Poxvirus Vectors The first clinical assessment of a viral vector vaccine for malaria was of NYVAC-Pf7 (2). This attenuated orthpoxvirus expressed three pre-erythrocytic antigens, CSP, TRAP/SSP2 and LSA-1, three blood-stage antigens, MSP1, AMA1 and SERA1, and a single sexual-stage antigen Pfs25. Antibody responses were induced but at low levels and some CTL responses were detectable with re-stimulated cells. Of the 35 sporozoitechallenged volunteers, one was completely protected and, overall there was a significant delay in time to patent parasitemia. Despite this partial success the sponsoring company (Aventis-Pasteur, now Sanofi Pasteur, Marcy-l’Etoile, France) decided not to develop this construct further. It remains unclear which antigen(s) contributed to the observed partial protection. Heterologous prime-boost regimens using a fowlpox and MVA vector have been assessed by the Oxford group using different inserts. Following studies of DNA-MVA vaccines encoding ME-TRAP fowlpox-MVA regimens were assessed clinically on the basis of the greater immunogenicity and efficacy of this new regimen in murine studies (40). In an initial pilot study two out of five vaccinees showed sterile protection (7). However, in larger numbers the efficacy was only slightly greater than with DNA-MVA, estimated as a 92% versus 82% reduction in liver parasite load (Fig. 1), an estimate based on real-time PCR analysis of sequentially measured parasite densities in the blood of challenged vaccinees and controls (68). Immunological analysis again showed poor antibody responses with fowlpox-MVA regimens, but good cellular immunogenicity. Direct comparison with DNA-MVA regimens showed that fowlpox-MVA induced a greater proportion of CD8 T cells but overall numbers of antigen specific cells were no higher (65). Phase I/IIa studies of the same vectors encoding the entire CSP gene were undertaken but showed lower immunogenicity than using the ME-TRAP insert and no protection (69). It remains unclear why CSP has yielded lower immunogenicity and efficacy than the TRAP antigen with both DNA-MVA and fowlpox-MVA regimens. More recent attempts to broaden the immune responses induced by fowlpox and MVA regimens using a six-antigen polyprotein insert (70) again showed poor immunogenicity and no efficacy against a sporozoite challenge in a phase I/IIa clinical trial (71, unpublished). Importantly, immunological analysis of ME-TRAP vaccinees who underwent sporozoite challenge found a significant correlation of both ex vivo and cultured interferon-g ELISPOT responses with protection against malaria (7,72). The latter assay appeared to correlate better but because the two responses are themselves correlated there was little power to distinguish the more relevant T-cell population. Consistent with the general difficulty in inducing sterile protection mediated by T cells, a level of about 1000 SFU per million appeared to be required at the peak of the response for sterile protection; however, lower responses were associated with partial protection manifest as a delay in time to parasitemia. The fowlpox-MVA vectors encoding ME-TRAP have progressed to phases I and IIb studies in Africa. In The Gambia safety was comparable to U.K. vaccinees but immunogenicity in adults was lower (64). Similarly in Kenyan adults and particularly in Kenyan children living in high transmission areas immunogenicity was lower than in the United Kingdom (73,74). A phase IIb trial was undertaken in about 400 one- to six-year-old children near Kilifi, Kenya and no efficacy was observed over 18 months of follow-up (75,76). Low immunogenicity, of an average of 100 SFU per million PBMCs correlated

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Figure 1 Efficacy of some candidate malaria vaccines measured by liver parasite burden reduction. The reduction in mean parasite liver burden was estimated as described in Ref. 68. Studies without a statistically significant protective effect are shown as 0% efficacy, although low-level protection may have been present without reaching significance in the sample studied. Source: From Ref. 82.

with malaria prevalence but not with helminthic infection, suggesting that an immunosuppressive effect of hyper-endemic malaria may interfere with vectored vaccine potency in young African children (77,78). Novel alternating vector immunization regimens were assessed in the Kenyan studies, for example, administering MVA then fowlpox and then MVA again, and these appeared to lead to enhanced numbers of resting memory cells compared with standard heterologous prime-boost regimes (73). As expected there was considerable inter-individual heterogeneity in immunogenicity of vectored regimens encoding both CSP and ME-TRAP inserts, and multifunctional T cells expressing both IL2 and interferon-g soon after vaccination were predictive of better memory. In The Gambia a novel phase II trial design to screen for the efficacy of pre-erythrocytic vaccines was evaluated (79). This used clearance of parasites with multidrug therapy in adults exposed to malaria. Daily blood sampling with real-time PCR analysis to detect time to the onset of parasitemia through natural exposure provides an opportunity to screen vaccines for some field efficacy in smaller numbers of volunteers than other trial designs.

Adenovirus Vectors The marked immunogenicity of adenovirus vectors in preclinical studies has led to the recent initiation of phase I/IIa clinical trials by several groups. The U.S. Navy—Genvec collaboration has initiated clinical trials of the standard Ad5 vector encoding AMA1 and CSP using a mixture of two vectors each encoding one antigen (80). To reduce the problem of high prevalences of anti-Ad5 antibodies in humans, especially in malaria-endemic areas, the company Crucell (Leiden, The Netherlands) has generated a CSP recombinant using the rare Ad35 serotype against which antibodies are less prevalent in humans. This is currently in phase I trials in the United States (81). A different approach is to use chimpanzee adenoviruses against which high-titer antibodies in humans are found only rarely. The Oxford group, in collaboration with the company Okairo`s

(Basel, Switzerland), is currently assessing the ME-TRAP insert in the AdCh63 vector (Fig. 2) with and without a heterologous boost with MVA. In a phase I trial, stronger T-cell and antibody immunogenicity have recently been observed than with previous vectored vaccine regimens employing ME-TRAP (Hill et al. unpublished). Both the U.S. Navy-Genvec and Oxford-Okairo`s groups aim to assess further constructs in the clinic in the near future, attempting to broaden the range of antigens targeted by this approach.

PROSPECTS There has been substantial progress in assessing the potential utility of vectored vaccines since their discovery in the mid1980s. Several periods of activity can be identified. Initially recombinant poxviruses were assessed and found to be modestly immunogenic for antibody induction, and to induce detectable cellular immunity. However, at this time, the lack of quantitative assays of cellular immunogenicity impaired assessments, as is well illustrated by the misconception that persisted until recently that many adjuvanted protein vaccines could compete with vectors for strong T-cell immunogenicity. In the mid- to late 1990s, extensive efforts to assess DNA vaccines led to the disappointing conclusion that their antibody immunogenicity in humans is generally very poor, and that induction of effector T cells rather modest. However, they have found a place in prime-boost regimens to induce T cells that can be boosted by viral vectors. Heterologous prime-boost regimens, using DNA and poxviral vectors, were tested in the clinic from 1999 and have shown stronger T-cell immunogenicity than single vectors, allowing a likely threshold of T-cell immunogenicity required for protection in humans to be identified (82). However, this revealed just how potent vaccines inducing cellular immunity will need to be to provide protection against malaria and emphasized the tendency for both DNA and poxvirus vaccines to induce CD4þ rather than CD8þ T cells.

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Figure 2 Phylogenetic tree of adenoviruses. Phylogenetic tree of human and chimpanzee adenoviruses, based on hexon sequences. ‘‘Hu’’ indicates a human virus and ‘‘Ch’’ a chimpanzee-derived virus. Three of these are currently in clinical trials as malaria vaccine vectors: the human viruses Ad5 and Ad35 and the chimpanzee-derived virus AdCh63. Source: Courtesy S. Gilbert, Oxford.

The greater capacity of adenoviruses to induce CD8þ than CD4þ responses and new strategies for avoiding antivector immunity has led to current excitement about the potential of adenoviral vectors as malaria vaccines. This is enhanced by the capacity of these vectors to induce better antibody responses than either DNA or poxviruses. Just how well this potential will translate into useful candidate vaccines should emerge in the next five years. With any new vaccine technology, safety is of paramount importance. Perhaps the greatest achievement of vectored approaches in the last 10 years (Table 1) has been the accumulation of considerable evidence that several vectors appear safe Table 1 Malaria Vaccine Vectors Assessed in Clinical Studies DNA

References

CSP ME-TRAP SSP2/TRAP Exp1 LSA-1 LSA-3 Poxviruses NVYAC Pf7 MVA ME-TRAP MVA CSO MVA Polyprotein FP9 ME-TRAP FP9 CSO FP9 Polyprotein Adenoviruses Ad5 CS Ad5 AMA1 Ad35 CS AdCh63 ME-TRAP

4, 66 5, 66 59 59 59 59 2 5, 7, 64, 66, 75 69, 66 71, unpublished 7, 75 69 71, unpublished Richie et al. unpublished Richie et al. unpublished 71, unpublished O’Hara et al. unpublished

Abbreviations: ME-TRAP, multiple epitope-thrombospondin-related adhesion protein; TRAP, thrombospondin-related adhesion protein; MVA, modified vaccinia Ankara; CSP, circumsporozoite protein.

for general use in humans, even though this statement must be tempered by the relatively modest samples sizes studied to date in most trials, and some anxiety generated by subgroup analyses of a recent HIV trial (83). Large numbers of malaria trials have revealed far fewer safety issues with vectored vaccines than with protein/adjuvant approaches. Further progress could be made by removing vaccines from the unhelpful ‘‘gene therapy’’ categorization used by some regulatory authorities, and by a more widespread understanding that vectors such as MVA are actually nonreplicating rather than replication-impaired in normal human tissues. A widespread misunderstanding is that exceptional potency must imply some replication capacity. It is likely that future generations of vector vaccine candidates will iteratively include improvements in vector design. Different strains of adenovirus differ markedly in potency and growth characteristics for reasons that are very poorly understood, and considerable activity is focused on generating chimeric viruses with better in vitro growth characteristics, improved potency, and less susceptibility to prevalent anti-vector immunity in humans. A parallel activity is to attempt to adjuvant vectors either with traditional adjuvants that would be mixed with the vaccines or with a new generation of ‘‘internal’’ adjuvants. Internal adjuvants represent gene products expressed with the antigen of interest from the viral vector that are designed to enhance immune responses to the antigen. The use of co-stimulatory molecules and toll-like receptor signaling pathways molecules appear promising for this application. Other major questions relate to the optimal immunization regimens for use of vectors in humans, particularly in heterologous prime-boost regimens. Most trials to date have used relatively short intervals of a month or two between the different vectors, but some preclinical data argue that longer intervals may be better. Although there are published data now on the safe and immunogenic use of vectors in young children,

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Figure 3 Quality of T-cell responses as assessed by polychromatic flow cytometry after various vectored vaccine regimens. The pie charts display the proportion of murine T cells, specific for a nonamer CD8 T-cell epitope in the P. berghei circumsporozoite protein, that contain 1, 2, or 3 of the cytokines interferon-g, TNF or IL2 after immunization. All vaccine vectors encode the ME-TRAP pre-erythrocytic insert (Table 1). (A) T-cell quality after single vector immunization. AdH5: the widely used human adenovirus serotype 5; AdC6, 7, 9, and AdCh63 are various chimpanzee adenoviral vectors; FP9 is a fowlpox vector. (B) T-cell quality after a booster immunization with the MVA vector encoding METRAP. The T cells are more polyfunctional, with more cells expressing three cytokines, after the heterologous vector boost. Abbreviations: ME-TRAP, multiple epitope-thrombospondin-related adhesion protein; MVA, modified vaccinia Ankara. Source: Ref. Reyes-Sandoval, unpublished data.

very little is known of safety and immunogenicity in infants, the major target population for malaria vaccines. Similarly, we need more data on how easily vectors expressing different antigens may be mixed without interference. For some DNA vaccines, particular antigens have been problematic in mixtures, but early impressions are that this is less of an issue with viral vectors. Of particular current interest is the quality of Tcell response induced by different vectors, as defined by the multiplicity of cytokines expressed by responding T cells (Fig. 3). MVA boosting has recently been found to shift the quality of T cells induced by adenoviruses to a more polyfunctional profile, expressing more cytokines, a feature suggested to be of more protective value. Much of the progress with new vaccine technologies in malaria has depended on the use of small-scale challenge studies with infectious mosquito bites. Now, with the feasibility of low dose challenge studies with blood-stage parasites (84,85), such phase IIa challenge studies may become of ever greater importance. With the increasing recognition that single component malaria vaccines are unlikely to generate the protective efficacy required for field deployment in the medium term, increasing effort is directed toward ‘‘building’’ a multicomponent vaccine formulation of iteratively increasing efficacy, through sequential evaluation of potential improvements. The opportunity to evaluate improved efficacy in small-scale human trials is rare in infectious diseases, and the malaria field should continue to avail of this advantage in evaluating new vaccine technologies.

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protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect Immun 2006; 74:5933–5942. Moorthy VS, Imoukhuede EB, Milligan P, et al. A randomised, doubleblind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults. PLoS Med 2004; 1:e33. Bejon P, Andrews L, Andersen RF, et al. Calculation of liver-toblood inocula, parasite growth rates, and preerythrocytic vaccine efficacy, from serial quantitative polymerase chain reaction studies of volunteers challenged with malaria sporozoites. J Infect Dis 2005; 191:619–626. Walther M, Thompson FM, Dunachie S, et al. Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein. Infect Immun 2006; 74:2706–2716. Prieur E, Gilbert SC, Schneider J, et al. A Plasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses. Proc Natl Acad Sci U S A 2004; 101:290–295. Porter DW, Thompson FM, Berthoud TK, et al. A human Phase IIa malaria challenge trial of a novel polyprotein malaria vaccine. Unpublished. Keating SM, Bejon P, Berthoud T, et al. Durable human memory T cells quantifiable by cultured enzyme-linked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol 2005; 175:5675–5680. Bejon P, Kai OK, Mwacharo J, et al. Alternating vector immunizations encoding pre-erythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur J Immunol 2006; 36:2264–2272. Bejon P, Mwacharo J, Kai OK, et al. Immunogenicity of the candidate malaria vaccines FP9 and modified vaccinia virus Ankara encoding the pre-erythrocytic antigen ME-TRAP in 1–6 year old children in a malaria endemic area. Vaccine 2006; 24:4709–4715. Bejon P, Mwacharo J, Kai O, et al. A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA METRAP among children in Kenya. PLoS Clin Trials 2006; 1:e29. Bejon P, Ogada E, Mwangi T, et al. Extended follow-up following a phase 2b randomized trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS ONE 2007; 2:e707. Bejon P, Keating S, Mwacharo J, et al. Early gamma interferon and interleukin-2 responses to vaccination predict the late resting memory in malaria-naive and malaria-exposed individuals. Infect Immun 2006; 74:6331–6338. Bejon P, Mwacharo J, Kai O, et al. The induction and persistence of T cell IFN-gamma responses after vaccination or natural exposure is suppressed by Plasmodium falciparum. J Immunol 2007; 179:4193–4201. Imoukhuede EB, Andrews L, Milligan P, et al. Low-level malaria infections detected by a sensitive polymerase chain reaction assay and use of this technique in the evaluation of malaria vaccines in an endemic area. Am J Trop Med Hyg 2007; 76:486–493. Doolan DL, Stewart VA. Status of malaria vaccine R&D in 2007. Malaria vaccines for the World 2007, September 17–19th, 2007, London, U K. Expert Rev Vaccines 2007; 6:903–905. Shott JP, McGrath SM, Pau MG, et al. Adenovirus 5 and 35 vectors expressing Plasmodium falciparum circumsporozoite surface protein elicit potent antigen-specific cellular IFN-gamma and antibody responses in mice. Vaccine 2008; 26:2818–2823. Hill AV. Pre-erythrocytic malaria vaccines: towards greater efficacy. Nat Rev Immunol 2006; 6:21–32. Hanke T. STEP trial and HIV-1 vaccines inducing T-cell responses. Expert Rev Vaccines 2008; 7:303–309. Sanderson F, Andrews L, Douglas AD, et al. Blood-stage challenge for malaria vaccine efficacy trials: a pilot study with discussion of safety and potential value. Am J Trop Med Hyg 2008; 78:878–883. Pombo DJ, Lawrence G, Hirunpetcharat C, et al. Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet 2002; 360:610–617.

71 Pre-erythrocytic and Asexual Erythrocytic Stage Whole-Organism Malaria Vaccines Stephen L. Hoffman, B. Kim Lee Sim, and Adam Richman Sanaria Inc., Rockville, Maryland, U.S.A.

Alberto Pinzon-Charry, Michael F. Good, and J. McCarthy Queensland Institute of Medical Research, Queensland, Australia

INTRODUCTION Subunit malaria vaccine candidates achieving high levels of protection have thus far proven difficult to develop. In contrast, strong protective immunity has been observed in humans and animals using the so-called whole-parasite approach. Immunization strategies targeting pre-erythrocytic malaria stages using radiation-attenuated sporozoites (in humans and animals) and genetically attenuated sporozoites (in mice) have been shown to result in sterile immunity. Immunization with asexual erythrocytic stage parasites followed by clearance with antimalarial chemotherapy results in delayed parasitemia. Here we review some of the developments that are advancing a whole-organism vaccine strategy against malaria. There have been promises of the development of malaria vaccines for more than 25 years. These hopes have been largely based on two sets of observations. First, immunization of volunteers with radiation-attenuated Plasmodium falciparum sporozoites (PfSPZs) induces immune responses that completely prevent infection upon challenge with fully virulent sporozoites (1–12). Second, several years of natural exposure and repeated infections with P. falciparum (Pf) lead (in surviving individuals) to an acquired immunity that does not prevent infection but reduces the density of parasitemia and the severity of clinical disease (13–15). On the basis of animal studies, it is thought that the protective immunity engendered by immunization with radiation-attenuated sporozoites is mediated primarily by T cells that recognize and eliminate parasite-infected hepatocytes and secondarily by antibodies against sporozoites that prevent hepatocyte invasion and normal parasite development (16–18). On the basis of passive transfer studies in humans (19–21), it has been thought that antibodies against asexual erythrocytic stage proteins are primarily responsible for the protective immunity induced by natural infections with Pf. However, multiple infections over several years are required to induce the range of antibodies required to suppress most strains of the parasite. There are also data supporting a role for T cell–mediated immunity against the asexual erythrocytic stages (22,23). While

the induction of an antibody response has been thought critical to vaccine development for asexual erythrocytic stages, little consideration has been given to developing a vaccine against these stages that would induce protection via a cell-mediated response. Despite the fact that exposure to the whole-parasite elicits such good immunity, until recently it was considered impossible to develop, license, and commercialize whole-parasite malaria vaccines. Thus, there have been attempts to understand which of the parasite proteins encoded by the more than 5300 genes in the Pf genome (24) are the targets of the protective immune responses, and to develop recombinant or synthetic subunit malaria vaccines that induce the required immune responses against the identified targets. Most of these efforts have focused on a single protein or at most several proteins or associated epitopes. These efforts are described in other chapters. In contrast, we are now focusing our efforts on wholeparasite approaches. Whole-organism vaccines have a long and successful history. Pioneering work by Jenner in the 18th century demonstrated the protective effect of vaccination with cowpox infection against smallpox (25). Numerous successful vaccines were subsequently developed, well before a satisfactory understanding of the biological mechanisms and protein and epitope targets of protective immunity was reached. These include the successful development and widespread deployment over the 20th century of attenuated vaccines against viruses (e.g., polio, measles, mumps rubella, yellow fever, Japanese Encephalitis), and bacteria [e.g., the Bacille Calmette–Gue´rin (BCG) vaccine for tuberculosis (26), cholera, and typhoid vaccines (27)]. In addition to live attenuated organisms, whole killed organism vaccines have been shown to be effective against numerous viral (polio, hepatitis A and B, rabies, influenza,) and bacterial diseases (pertussis, typhoid, plague, and cholera). In fact, whole-organism vaccines (both live attenuated and killed) represent 75% of currently licensed formulations (28), attesting to their safety and efficacy.

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For malaria, however, whole-parasite vaccines have been viewed as impractical to develop because of the complex life cycle of the parasite as well as logistical concerns relating to difficulties in large-scale production. Recent advances in bioengineering, basic parasitology, and entomology have enabled scientists to develop means to overcome these obstacles. In this chapter, we describe efforts to develop an attenuated PfSPZ vaccine and a killed asexual Pf erythrocytic stage whole-parasite vaccine.

PRE-ERYTHROCYTIC STAGE WHOLE-PARASITE MALARIA VACCINES Metabolically Active, Nonreplicating (Attenuated) Plasmodium falciparum Sporozoite Vaccine Background. In 2002, we updated and summarized the world’s published literature on immunizing humans by the bite of Anopheles mosquitoes infected with PfSPZ (12). The data were striking. Fourteen volunteers were immunized by the bite of greater than 1000 Pf-infected, irradiated mosquitoes. When challenged by the bite of five nonirradiated Pf-infected mosquitoes 2 to 10 weeks after their final immunization, 13 of the 14 volunteers (93%) were protected against developing asexual erythrocytic stage infection with Pf. Six of the volunteers were rechallenged a total of 15 times within 2 to 10 weeks of final immunization, and all six volunteers were entirely protected in all 15 challenges (100%). In addition, six volunteers were challenged 23 to 42 weeks (at weeks 23, 36, 36, 39, 41, and 42) after final immunization and five of the six volunteers were protected. In total, there were 35 challenges in these volunteers and there was complete protection against Pf infection in 33 of the 35 challenges (94%). These challenges were done primarily with isolates of Pf identical to the parasites that had been used to immunize the volunteers. In four volunteers seven challenges were also done with heterologous parasite strains. The parasites used for immunization and challenges originated from geographically distinct locations. Nonetheless, there was protection in all of the volunteers who received heterologous challenge (100%). When volunteers were immunized with less than 1000 immunizing bites, and then challenged with homologous isolates, there was protection in only 5 of 15 challenges (33%). From the time of first publication of results on immunizing volunteers by the bite of irradiated mosquitoes infected with PfSPZs in the early 1970s (1–8), there was essentially universal recognition that immunization with radiationattenuated PfSPZs proved the principle that it was possible to immunize humans against malaria, and that this form of immunization was a gold standard for malaria vaccine development. However, there was also essentially universal consensus that it was impractical to consider developing an attenuated PfSPZ vaccine. It was believed to be impossible to produce and deliver adequate quantities of aseptic, purified, well-characterized, stable PfSPZs that meet regulatory and cost of goods requirements. Interestingly, there was little concern regarding the potential safety of such an attenuated PfSPZ vaccine. T cells from volunteers immunized by the bite of irradiated PfSPZinfected mosquitoes were used to try to determine the immune responses and target antigens of such immune responses that were responsible for the protective immunity. The hope was that these findings would lead to the development of an effective subunit vaccine (29–39).

Sanaria was founded to overcome the perceived obstacles and to develop and commercialize an attenuated PfSPZ vaccine. At the outset, we thought that the chance of success was good for several reasons (40). The immunogen, attenuated PfSPZs, was already known to be highly protective. Thus, development of the vaccine was a bioengineering and applied parasitology and entomology challenge, not a molecular biology and immunology discovery problem. It involved manufacturing a vaccine for the first time in mosquitoes and controlling all of the elements of the manufacturing process. Since a large, profitable traveler’s market for the vaccine was anticipated, we created a plan for raising the estimated $0.5 to $1.0 billion required to develop, register, and deploy the vaccine to benefit the primary target population, infants in sub-Saharan Africa. The first attenuated PfSPZ vaccine entered clinical trials in spring of 2009. This has required several years of research, followed by a year of process development, followed by establishment of a manufacturing capability and finally by establishment of a clinical development plan. Research. When Sanaria was founded, there were three major questions that had to be addressed (40). Could one

1. 2. 3.

administer the vaccine by a route that was clinically appropriate? produce adequate quantitites of radiation-attenuated Plasmodium falciparum sporozoites (PfSPZ)? manufacture a PfSPZ vaccine meeting regulatory and cost of goods requirements?

Administer the vaccine by a clinically appropriate route. One of the first studies conducted by Sanaria addressed whether immunization and protection of rodents could be achieved through administration of IrrPySPZ through a nonintravenous (non-IV) route. IrrPySPZs were isolated from infected Anopheles stephensi mosquitoes by gradient density centrifugation. Two groups of six-week-old BALB/c mice were immunized with IrrPySPZ three times at two-week intervals. The IrrPySPZs were administered by either IV or subcutaneous (SC) routes using what was then considered the standard total dosage regimen. The same vaccine doses were used for both routes of administration. The first injection was 50,000 IrrPySPZs, and the second and third injections were 30,000 IrrPySPZs each, for a total of 110,000 IrrPySPZs per mouse. Mice were challenged with an IV injection of 100 nonattenuated PySPZ two weeks following the third injection of vaccine. The mice were monitored daily for infection (blood smears examined for parasitemia) for 14 days following challenge. One hundred percent of the mice in each group were protected against the development of blood-stage infection (Table 1) and all of the control, nonimmunized mice developed infections (Hoffman SL and Sedegah M, unpublished). Thus, Table 1 Protection Against Plasmodium yoelii Sporozoite Challenge in Mice by Immunization with Radiation-Attenuated P. yoelii Sporozoite Vaccine Administered by IV or SC Injection

Group

Number of mice challenged

Number of mice protected/ challenged

Percentage of mice protected (%)

Controls IV SC

8 7 8

0/8 7/7 8/8

0 100 100

Abbreviations: IV, intravenous; SC, subcutaneous.

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vaccination by either IV or SC routes, at this dose level and schedule, induced protection against IV administration (challenge) of freshly dissected, nonattenuated PySPZs. In a subsequent experiment, 10 BALB/c mice were immunized SC with three doses of IrrPySPZ that had been dissected by hand from salivary glands (9000 IrrPySPZs, 3000 IrrPySPZs, and 3000 IrrPySPZ, a total of 15,000 IrrPySPZ) administered at two-week intervals. When challenged two weeks after the last dose of IrrPySPZ, 8 of 10 mice were protected, and all naı¨ve control mice developed parasitemia. These experiments demonstrated that mice could be effectively immunized by administration of IrrPySPZ by the SC route. Subsequently, Sanaria scientists have been able to demonstrate that 100% of mice can be protected by immunization with previously cryopreserved IrrPySPZ administered by the SC or intradermal (ID) route. These studies provide a foundation for the next step, which is to determine the optimal route of administration in humans in a clinical trial (see below). Produce adequate quantitites of PfSPZ. Until the number of PfSPZ per dose and the dosage regimen have been established, it will be impossible to know what resources will be required to produce adequate quantities of sporozoites. Producing adequate numbers of PfSPZ-infected mosquitoes will not be a rate-limiting step. When Sanaria was founded we wondered whether dissection of salivary glands from mosquitoes might be limiting. Currently, a six-person Sanaria dissection team can remove the salivary glands from approximately 500 mosquitoes per hour (Fig. 1). When we establish the final dose of SanariaTM PfSPZ vaccine and the dosage regimen, we will be able to establish the hourly output of PfSPZ vaccine required from the dissection team. However, we are now confident that it is feasible to produce adequate quantities of the PfSPZ vaccine.

Manufacture PfSPZ vaccine meeting regulatory requirements. Such a vaccine must, at a minimum, be (i) free of contaminating pathogens, (ii) free of significant amounts of mosquito-derived material, (iii) completely attenuated (i.e., safe), and (iv) potent (i.e., capable of eliciting a protective immune response). Sanaria scientists have now developed methodologies, equipment, and standard protocols for producing the live attenuated PfSPZ vaccine by an aseptic process that yields pathogen-free sporozoites as determined by standard FDA-mandated assays. Methods to remove contaminating mosquito material from the PfSPZ and an assay to

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measure such material have also been developed. Working with the U.S. National Institute of Standards (NIST), Sanaria has developed a dosimetry-based monitoring system that measures the minimum and maximum dose of irradiation that each mosquito receives. Having established the minimum dose of irradiation that attenuates all parasites, the results of the dosimetric monitoring together with an in vitro attenuation assay developed by Sanaria ensures that all sporozoites are adequately attenuated. Finally, Sanaria has established an in vitro potency assay. With the methodologies established to produce PfSPZ vaccine that is free of pathogens, uncontaminated by significant amounts of mosquito material, adequately attenuated, and potent, it remained to implement a current Good Manufacturing Practices (cGMP)-compliant manufacturing process. Process development. Component procedures to produce the vaccine were studied with considerations given to yields, quantities, quality, speed, timing, adaptability to scale, and suitability for conformance manufacture under GMPs. These component procedures were then integrated to generate a single coordinated flow of process that included: producing aseptic mosquitoes and gametocyte cultures, feeding of mosquitoes, maintaining infected aseptic mosquitoes, irradiating infected mosquitoes to attenuate sporozoites, harvesting of PfSPZ from the mosquitoes, removing salivary gland material from the harvested sporozoites without a major reduction in yield or potency, formulating bulk PfSPZ preparations, and cryopreserving PfSPZ. In-process assays that monitor the integrity and performance of the process at multiple steps during production were developed and implemented. Release assays that characterize and describe bulk PfSPZ preparations and final PfSPZ vaccine were also developed and implemented. More than 10 integrated production campaigns designed to solidify and document the capability of our manufacturing process were conducted during 2006. Quality Systems and Manufacturing teams together with external consultants established our standard operating procedures and batch records required for cGMP manufacture.

Manufacturing under current Good Manufacturing Practices. PfSPZ vaccine lots for preclinical studies in support of the Investigational New Drug application. After establishing the manufacturing process and associated documentation, Sanaria conducted multiple production campaigns in 2007 to manufacture and release PfSPZ vaccine lots for preclinical toxicology and immunology studies in support of an Investigational New Drug (IND) application. The production campaigns were successfully completed and several lots were released for use in IND-enabling studies.

SanariaTM PfSPZ vaccine manufactured for clinical trials. Sanaria’s initial manufacturing efforts produced

Figure 1 The first stage of Plasmodium falciparum sporozoite isolation: dissection of salivary glands from mosquitoes.

vaccine lots suitable for preclinical IND-enabling studies. The existing facility, however, was deemed unsuitable for the manufacture of PfSPZ vaccine intended for clinical trials. Indeed, Sanaria’s production facility was described as occupying a ‘‘dismal strip mall in Rockville, MD’’ (41). In partnership with the PATH Malaria Vaccine Initiative, a grant was obtained from the Bill and Melinda Gates Foundation to support vaccine development efforts. A portion of these funds was used to build the world’s first facility for manufacturing a live attenuated malaria vaccine. Clinical production campaigns were conducted from March through July 2008. All vaccine lots were successfully tested and released for clinical studies.

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Clinical development plan. The first clinical trial of the radiation-attenuated SanariaTM PfSPZ vaccine was initiated in spring of 2009 at the Naval Medical Research Center and the University of Maryland Center for Vaccine Development, both in Maryland, U.S.A. This is a Phase 1 safety, immunogenicity, and protective efficacy study. The primary goal is to establish that the vaccine is safe, well tolerated, and immunogenic. Because volunteers can be safely infected with Pf by the bite of mosquitoes transmitting live, nonattenuated sporozoites (42), the protective efficacy of the vaccine will also be assessed. This is a dose escalation study using two routes of administration (SC and ID). It is anticipated that subsequent studies will address multiple potential variables including the route and method of administration, number of doses, interval between doses, volume of administration, and site of administration. In addition, the longevity of protection, protection against different geographic isolates of Pf, and protection against Plasmodium vivax will be determined in experimentally infected, malaria-naive volunteers. Once vaccine safety has been demonstrated in clinical studies performed in the United States, it is anticipated that trials will be conducted in sub-Saharan Africa. The goal of these trials will be first to demonstrate that the vaccine is safe and immunogenic in malaria-exposed adults and then move rapidly to assessment of the vaccine safety, immunogenicity, and efficacy in the populations that suffer the most from malaria caused by Pf. The primary target population is the 25 million babies born annually in sub-Saharan Africa. Preadolescent girls are another important target population because malaria during pregnancy is associated with increased maternal morbidity and mortality, spontaneous abortion, and low birth weight infants who are at increased risk of dying in the first year of life. Summary. In 2003, Sanaria scientists reappraised the potential impact of a metabolically active, nonreplicating PfSPZ vaccine, and systematically outlined the obstacles to producing such a vaccine (40). Six years later, significant progress has been made in overcoming these obstacles, enabling the manufacture of clinical lots of an PfSPZ vaccine and the initiation of safety, immunogenicity, and protective efficacy studies in volunteers (phase 1 clinical trials).

ASEXUAL ERYTHROCYTIC STAGE WHOLEPARASITE MALARIA VACCINES Plasmodium falciparum Asexual Erythrocytic Stage Vaccines Live (Nonattenuated) and Killed Parasite with Adjuvant Background. Whole-parasite erythrocytic vaccines have usually relied on immunization through repetitive infection with blood-stage parasites. Protocols for the treatment of neurosyphilis, whereby patients were repeatedly challenged with blood infections could be regarded as the first clear demonstration of the potential for whole-parasite blood-stage vaccines. While these studies were only observational, they indicated that strain-specific immunity developed following several rounds of infection (43,44). These findings spurred interest in investigating the effects of immunization with whole killed blood-stage parasites. However, studies using killed bloodstage P. vivax (45) failed to induce any significant protection and this strategy was not pursued further. The subsequent success of radiation-attenuated sporozoites to induce protection in mice and humans (see above) led other groups to study immunization with radiation-attenuated

blood-stage parasites. In the mouse model, immunization with the rodent parasites Plasmodium berghei or P. yoelii by multiple intravenous injections of large doses of irradiated blood-stage parasites was shown to protect against lethal challenge (46). Similarly, protective immunity was observed in other models of infection, including rats (47) and monkeys (48). However, this approach had limited potential due to difficulties related to the large-scale production of the immunogen. A major breakthrough occurred in 1976 with the report by Trager and Jensen (49) of the in vitro culture of the blood stage of Pf. This was followed by a series of studies demonstrating the feasibility of producing subunit antigens (50,51) for vaccines targeting blood, liver, and mosquito stages of the parasite life cycle (52). Several such candidate vaccines have been clinically tested though only one, RTS,S, has progressed to a large-scale phase III clinical trial (53). Impediments to the development of a successful subunit vaccine include an incomplete knowledge of the critical targets of a protective immune response, overcoming antigenic polymorphism, evoking a robust and long-lasting immune response, and overcoming major histocompatibility complex (MHC) haplotype restriction. It is notable that a critical component of the only subunit vaccine to reach phase III clinical trial is the potent adjuvant, ASO2A, containing the immunostimulants monophosphoryl lipid A and fraction 21 from the plant Quillaja saponaria. Without this adjuvant, the circumsporozoite subunit vaccine does not lead to significant protection. As demonstrated by studies of malaria therapy for syphilis, and by immuno-epidemiological studies in endemic settings, it is known that repetitive infections with Pf are necessary to provide protection against the clinical illness associated with malaria. While a protective antibody response is known to be important, a number of rodent studies have indicated that cell-mediated immunity (CMI), mediated primarily by CD4þ T cells, may also play an important role in protection.

Live (nonattenuated) whole-parasite approach (human data). The data supporting the potential importance of CMI responses have focused current efforts on developing immunization strategies that result in a long-lasting and protective CMI response targeting as many parasite antigens as possible. Recently a ‘‘whole-organism’’ approach based on immunization of humans with very low doses of viable blood-stage parasites was explored (22). These studies demonstrated that repeated infections of naı¨ve human volunteers with ultra-low doses of blood-stage Pf parasites, followed by drug cure after six to eight days (before the onset of clinical symptoms) resulted in the induction of strong parasite-directed CMI responses in the absence of significant antibody responses (Fig. 2). In rodent models, a similar immunization strategy has been shown to induce protection against homologous and heterologous Plasmodium infection challenge (23). However, such protocols would be extremely difficult to deploy in a field setting and are thus not suitable for development as an immunization strategy.

Killed whole parasite with adjuvant approach (animal data). Recent work by Su et al. (54) suggests an alternative immunization strategy. The investigation showed in a rodent model that large doses of whole-parasite antigen (parasite extract) adjuvanted in interleukin (IL)-12 or CpG DNA could completely protect against an otherwise lethal challenge. These results demonstrate that solid protection can

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Figure 2 Cell-mediated immunity responses of volunteers. Lymphoproliferation and cytokine production, in response to schizont-enriched P. falciparum–parasitised red cells, of peripheral blood mononuclear cells before study (gray bars) and after exposure to subpatent infections (black bars).

be achieved in a rodent model with ‘‘killed parasite.’’ Because high parasite loads can lead to apoptosis of effector T cells, the approach of Su et al. (54) has been adapted and used to investigate the effectiveness of immunization using very low doses of killed parasite immunogen adjuvanted with CpG DNA (55). The preliminary results confirm significant reductions in the levels of parasitemia detected in mice immunized with high doses of parasites (>106) plus adjuvant following infection challenge with virulent parasites. Furthermore, survival from otherwise lethal challenge is uniform among immunized mice. Immunization with low doses of immunogen (103

parasites) also protects 100% of animals, while immunization with ultralow doses (102 parasites) affords 60% protection. Just as importantly, low-dose immunization appears to confer longterm protection (>12 weeks, 100% survival), as well as vigorous T-cell responses against both homologous and heterologous parasites (Pinzon-Charry et al., unpublished data). These observations suggest that robust heterologous protective immunity might be induced in humans using a lowdose ‘‘dead’’ formulation of Pf adjuvanted with CpG and alum. While there have been relatively few clinical trials in humans of vaccines that have included a CpG adjuvant, it is apparent that

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such adjuvants induce a qualitatively different immune response to that seen with the major adjuvant used in humans, namely alum. For example, immunization with hepatitis B surface antigen adjuvanted with CpG resulted in an earlier appearance and significantly higher titer of a hepatitis B surface antibody compared with the standard vaccine that included an alum adjuvant (56). Indeed, most CpG-vaccinated subjects developed protective levels of anti-HBs IgG within just two weeks of the first dose of vaccine. In a second study, an equivalent T-cell response of PBMCs was observed from human subjects immunized with either full-dose vaccine or a 10-fold lower dose of killed influenza vaccine adjuvanted with CpG (57).

Killed whole parasite with adjuvant approach (toward clinical trials). The rationale supporting a wholeparasite formulation including a potent CpG adjuvant as a malaria vaccine for humans includes the following considerations: circumvention of the need to precisely identify which (and perhaps which combination of) parasite antigen(s) is/are necessary for inclusion in a malaria vaccine; potentially bypassing the problem of MHC restriction imposed by a simpler subunit vaccine, which inevitably contains a more restricted set of potential antigens; addition of a novel adjuvant that induces a potent T helper 1 (TH1) bias in the evoked immune response; the heterologous protection against different malaria strains and species observed in the rodent model, suggesting that this approach might overcome the problems of antigenic polymorphism among Pf strains, and antigenic variation under immune pressure during in vivo infection; enhanced feasibility of large-scale vaccine production if only a low dose of parasite immunogen is required. For example, if 104 parasite-infected erythrocytes are required for vaccination, a single unit of blood containing approximately 109 infected red cells would provide sufficient blood for one hundred thousand doses of vaccine. The research issues demanding attention before such a vaccine can be developed include the following:

1.

2.

3.

The challenge of demonstrating disease attenuation as an end point for a vaccine that is unlikely to prevent parasitemia, but rather act by protecting against uncontrolled blood-stage parasite escalation. The design of any clinical trials will therefore require the ascertainment of a variety of clinical end points. It is worth noting that this issue also holds for subunit blood-stage vaccines currently in phase 1 clinical trials (AMA1, MSP2, etc.). While a significant body of data establishes the safety of CpG-adjuvanted vaccines in adults, this is less the case for children. Given the potency of this adjuvant in inducing a TH1 response, safety will need to be established. Establishing that this approach does not induce clinically significant allo-immunization to red cell antigens.

Summary and future directions for a whole-parasite asexual erythrocytic stage approach. The disappointing results of malaria vaccine clinical trials to date have forced a reappraisal of current strategies. Alternative approaches are now being evaluated. As the malaria parasite is proving a difficult target for vaccine development, the exploration of several different strategies offers better hope for the identification of a successful vaccine. The obstacles to manufacturing whole-organism asexual erythrocytic stage vaccines should be reconsidered in light of demonstrated protective efficacy in rodent models and intriguing results of low-dose immunization protocols in a human clinical trial. In addition to further

proof-of-concept studies, concerns related to large-scale commercial production and consistent formulation of such a vaccine must be addressed. The work on development of the attenuated PfSPZ vaccine establishes a foundation and precedents on which efforts to produce a whole-organism, asexual erythrocytic stage Pf vaccine can now be based.

REFERENCES 1. Clyde DF, Most H, McCarthy VC, et al. Immunization of man against sporozoite-induced falciparum malaria. Am J Med Sci 1973a; 266:169–177. 2. Clyde DF, McCarthy VC, Miller RM, et al. Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am J Med Sci 1973b; 266:398–401. 3. Clyde DF, McCarthy VC, Miller RM, et al. Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. Am J Trop Med Hyg 1975; 24:397–401. 4. Clyde DF. Immunity to falciparum and vivax malaria induced by irradiated sporozoites: a review of the University of Maryland studies, 1971–75. Bull World Health Organ 1990; 68:9–12. 5. Rieckmann KH, Carson PE, Beaudoin RL, et al. Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans R Soc Trop Med Hyg 1974; 68:258–259. 6. Rieckmann KH, Beaudoin RL, Cassells JS, et al. Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria. Bull World Health Organ 1979; 57:261–265. 7. Rieckmann KH. Human immunization with attenuated sporozoites. Bull World Health Organ 1990; 68:13–16. 8. McCarthy VC, Clyde DF. Plasmodium vivax: correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Exp Parasitol 1977; 41(1):167–171. 9. Herrington D, Davis J, Nardin E, et al. Successful immunization of humans with irradiated sporozoites: humoral and cellular responses of the protected individuals. Am J Trop Med Hyg 1991; 45:539–547. 10. Edelman R, Hoffman SL, Davis JR, et al. Long-term persistence of sterile immunity in a volunteer immunized with X-irradiated Plasmodium falciparum sporozoites. J Infect Dis 1993; 168:1066–1070. 11. Egan JE, Hoffman SL, Haynes JD, et al. Humoral immune responses in volunteers immunized with irradiated Plasmodium falciparum sporozoites. Am J Trop Med Hyg 1993; 49:166–173. 12. Hoffman SL, Goh LM, Luke TC, et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis 2002; 185(8): 1155–1164. 13. Beadle C, McElroy PD, Oster CN, et al. Impact of transmission intensity and age on Plasmodium falciparum density and associated fever: implications for malaria vaccine trial design. J Infect Dis 1995; 172:1047–1054. 14. Baird JK. Host age as a determinant of naturally acquired immunity to Plasmodium falciparum. Parasitol Today 1995; 11:105–111. 15. Aponte JJ, Menendez C, Schellenberg D, et al. Age interactions in the development of naturally acquired immunity to Plasmodium falciparum and its clinical presentation. PLoS Med 2007; 4:1259–1267. 16. Schofield L, Villaquiran J, Ferreira A, et al. Gamma interferon, CD8þ T cells and antibodies required for immunity to malaria sporozoites. Nature 1987; 330:664–666. 17. Weiss WR, Sedegah M, Beaudoin RL, et al. CD8þ T cells (cytotoxic/ suppressors) are required for protection in mice immunized with malaria sporozoites. Proc Natl Acad Sci U S A 1988; 85:573–576. 18. Doolan DL, Hoffman SL. The complexity of protective immunity against liver-stage malaria. J Immunol 2000; 165:1453–1462. 19. McGregor IA. The passive transfer of human malarial immunity. Am J Trop Med Hyg 1964; 13(suppl):237–239. 20. Sabchareon A, Burnouf T, Ouattara D, et al. Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria. Am J Trop Med Hyg 1991; 45:297–308.

Chapter 71: Whole-Organism Malaria Vaccines 21. Gysin J, Moisson P, Pereira da Silva L, et al. Antibodies from immune African donors with a protective effect in Plasmodium falciparum human infection are also able to control asexual blood forms of the parasite in Saimiri monkeys. Res Immunol 1996; 147:397–401. 22. Pombo DJ, Lawrence G, Hirunpetcharat C, et al. Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet 2002; 360:610–617. 23. Elliott SR, Kuns RD, Good MF. Heterologous immunity in the absence of variant-specific antibodies after exposure to subpatent infection with blood-stage malaria. Infect Immun 2005; 73: 2478–2485. 24. Gardner MJ, Hall N, Fung E, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002; 419: 498–511. 25. Barquet N, Domingo P. Smallpox: the triumph over the most terrible of the ministers of death. Ann Intern Med 1997; 127:635–642. 26. Calmette A. What is the importance of tuberculosis immunisation with BCG vaccine for the fight against tuberculosis? Dtsch Med Wochenschr 1931; 57:932–933. 27. Ditthorn F, Loewenthal W. The technology of cholera and typhus vaccine production in general. Dtsch Med Wochenschr 1915; 41:1006–1008. 28. The Jordan Report. 20th Anniversary. Accelerated development of vaccines. National Institute of Allergy and Infectious Diseases, NIH, Bethesda, 2002. 29. Malik A, Egan JE, Houghten RA, et al. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. Proc Natl Acad Sci U S A 1991; 88:3300–3304. 30. Wizel B, Houghten RA, Parker K, et al. Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum surface sporozoite protein 2. J Exp Med 1995; 182:1435–1445. 31. Wizel B, Houghten R, Church P, et al. HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J Immunol 1995; 155:766–775. 32. Krzych U, Lyon JA, Jareed T, et al. T lymphocytes from volunteers immunized with irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens. J Immunol 1995; 155:4072–4077. 33. Doolan DL, Hoffman SL, Southwood S, et al. Degenerate cytotoxic T cell epitopes from P. falciparum restricted by HLA-A and HLA-B supertypes alleles. Immunity 1997; 7:97–112. 34. Doolan DL, Southwood S, Chesnut R, et al. HLA-DR-promiscuous T cell epitopes from Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple HLA class II alleles. J Immunol 2000; 165:1123–1137. 35. Nardin EH, Herrington DA, Davis J, et al. Conserved repetitive epitope recognized by CD4þ clones from a malaria-immunized volunteer. Science 1989; 246:1603–1606. 36. Nardin EH. T cell responses in a sporozoite-immunized human volunteer and a chimpanzee. Immunol Lett 1990; 25:43–48. 37. Nardin EH, Nussenzweig RS, Altszuler R, et al. Cellular and humoral immune responses to a recombinant P. falciparum CS protein in sporozoite-immunized rodents and human volunteers. Bull World Health Org 1990; 68(suppl):85–87. 38. Moreno A, Clavijo P, Edelman R, et al. Cytotoxic CD4þ T cells from a sporozoite-immunized volunteer recognize the Plasmodium falciparum CS protein. Int Immunol 1991; 3:997–1003.

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39. Moreno A, Clavijo P, Edelman R, et al. CD4þ T cell clones obtained from Plasmodium falciparum sporozoite-immunized volunteers recognize polymorphic sequences of the circumsporozoite protein. J Immunol 1993; 151:489–499. 40. Luke TC, Hoffman SL. Rationale and plans for developing a nonreplicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 2003; 206:3803–3808. 41. Finkel M. Bedlam in the blood: malaria. Natl Geogr Mag 2007: 32–67. 42. Epstein JE, Rao S, Williams F, et al. Safety and clinical outcome of experimental challenge of human volunteers with Plasmodium falciparum-infected mosquitoes: an update. J Infect Dis 2007; 196:145–154. 43. O’Leary PA. Treatment of neurosyphilis by malaria—report on the three years observation of the first one hundred patients treated. JAMA 1927; 89:95–100. 44. O’Leary PA, Welsh AL. Treatment of neurosyphilis with malaria—observations on nine hundred and eightyfour cases in the last nine years. JAMA 1933; 101:498–501. 45. Heidelberger M, Prout C, Hindle JA, et al. Studies in human malaria: III. An attempt at vaccination of paretics against bloodborne infection with Pl. vivax. J Immunol 1946; 53:109–112. 46. Waki S, Yonome I, Suzuki M. Plasmodium yoelii: induction of attenuated mutants by irradiation. Exp Parasitol 1986; 62:316–321. 47. Wellde BT, Sadun EH. Resistance produced in rats and mice by exposure to irradiated Plasmodium berghei. Exp Parasitol 1967; 21:310–324. 48. Wellde BT, Diggs CL, Anderson S. Immunization of Aotus trivirgatus against Plasmodium falciparum with irradiated blood forms. Bull World Health Organ 1979; 57(suppl 1):153–157. 49. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976; 193:673–675. 50. Kemp DJ, Coppel RL, Cowman AF, et al. Expression of Plasmodium falciparum blood-stage antigens in Escherichia coli: detection with antibodies from immune humans. Proc Natl Acad Sci U S A 1983; 80:3787–3791. 51. Ellis J, Ozaki LS, Gwadz RW, et al. Cloning and expression in E. coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi. Nature 1983; 302:536–538. 52. Epstein JE, Giersing B, Mullen G, et al. Malaria vaccines: are we getting closer? Curr Opin Mol Ther 2007; 9:12–24. 53. Alonso PL, Sacarlal J, Aponte J, et al. Duration of protection with RTS,S/AS02A malaria vaccine in prevention of Plasmodium falciparum disease in Mozambican children: single-blind extended follow-up of a randomised controlled trial. Lancet 2005; 366:2012–2018. 54. Su Z, Tam M-F, Jankovic D, et al. Vaccination with novel immunostimulatory adjuvants against blood-stage malaria in mice. Infect Immun 2003; 71:5178–5187. 55. Pinzon-Charry A, Anderson V, McPhun V, et al. Malaria vaccines: new hope in old ideas. Drug Discov Today Therap Strategies 2006; 3:167–171. 56. Cooper CL, Davis HL, Morris ML, et al. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study. J Clin Immunol 2004; 24:693–701. 57. Cooper CL, Davis HL, Morris ML, et al. Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine. Vaccine 2004; 22:3136–3143.

72 Vaccines Against Leishmania Farrokh Modabber Drugs for Neglected Diseases Initiative (DNDi), Geneva Switzerland; Centre for Research and Training in Skin Diseases and Leprosy (CRTSDL), Tehran University of Medical Sciences, Tehran, Iran, & London School of Hygiene and Tropical Medicine, London, U.K.

Rhea Coler and Steven G. Reed Infectious Disease Research Institute, Seattle, Washington, U.S.A.

INTRODUCTION More than a dozen identified and characterized species of the genus Leishmania cause diseases ranging from simple selfhealing cutaneous lesions to debilitating and lethal (if untreated) visceral leishmaniasis (VL) known as kala-azar (Table 1). Other less frequent forms are mucosal leishmaniasis (ML) a highly disfiguring disease of oral and nasal cavities, and diffuse cutaneous leishmaniasis (DCL) with hundreds of nodular lesions spread over the body. Self-limiting visceral infections with some Leishmania—L. infantum (1), L. donovani (2), and possibly L. tropica (3)—may also occur. These asymptomatic individuals as well as cured cases would later develop disease if their immune responses are depressed by drugs or human immunodeficiency virus (HIV) infection, indicating nonsterile immunity following cure (4). Persistence of parasites in humans and in resistant mouse strains long after recovery of the initial lesion has been documented (5,6) and leishmaniasis recidivans (reappearance of new satellite lesions) around the original healed lesion is occasionally seen with L. tropica infection. In this respect, leishmaniasis is considered as an opportunistic infection (7). All forms of leishmaniasis are naturally transmitted by the bites of more than 70 different species of female sandflies either from infected humans (anthroponotic leishmaniasis) or from infected animals (zoonotic). The life cycle in the sandfly vector is confined to the alimentary tract and involves replication as extracellular flagellated promastigotes in the posterior midgut, differentiation to an infectious, nondividing metacyclic promastigote stage in the anterior gut, and inoculation into the skin of low numbers (100–1000) of metacyclics when the infected sandfly seeks another blood meal. Parasites that are inoculated into the skin are taken up by macrophages, and produce a spectrum of chronic diseases. Despite the incredible diversity of parasite and vector species, the establishment of Leishmania infection in the mammalian host involves the parasitization of macrophages in the skin and persistence and replication of nonflagellated amastigote-stage parasites in the phagolysosome of these host cells. While their presence in dendritic cells (DCs), neutrophils, and even fibroblasts has been described, there is no evidence that amastigotes can actively replicate in a cell other than a macrophage. Macrophages possess primary defense mechanisms, including activation of macrophage oxidative metabolism and synthesis and

release of arachidonic acid metabolites, that are induced by the attachment and engulfment of microbial agents. The major source of reactive oxygen intermediates (ROIs) in macrophages is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is a multimeric enzyme complex. Once assembled, the oxidase transfers an electron from NADPH to molecular oxygen, producing O 2
—which can dismutate to hydrogen peroxide (H2O2). ROI act together with reactive nitrogen intermediates (RNI), derived from nitric oxide, in the early stage of intracellular infection to regulate both tissue recruitment of mononuclear inflammatory cells and the initial extent of microbial replication. Previous studies have shown that RNI alone are necessary and sufficient for control of visceral infection, and although mature granulomas have traditionally been associated with control of such infections, these structures fail to limit intracellular parasite replication in the absence of iNOS (8). During the early establishment of infection in the skin and lymphoid organs, Leishmania produce multiple effects on macrophage and DC functions that inhibit their innate anti-microbial defenses and impair their capacity to initiate T helper 1 cell immunity. Generally, there is a good association between the organism and type of disease it produces in humans however, L. tropica usually the causative agent of anthroponotic CL (ACL) may cause VL as was seen in U.S. soldiers returning from the Middle East (3) and L. infantum the causative agent of zoonotic VL can cause CL (9). Approximately 350 million people are believed to be at risk of infection and the annual incidence of new cases is about two million, mostly in children and young adults (1.5 million CL and the rest VL). Infection is more common in men than in women, but this may reflect increased exposure to sandflies. Although disease occurs irrespective of age, children aged one to four years are particularly at risk of infection in the Mediterranean regions, and childhood infection may account for more than half of all cases in some of these countries. The current estimated prevalence is 12 million distributed in 88 countries (10). These figures do not include epidemics, which can claim the lives of tens of thousands individuals, eliminate communities and cause massive migration (11). The burden of disease expressed in disability-adjusted life years (DALYs) is estimated to be almost 2 million (Table 1) (12). With resistance to first line drugs (antimonials) up to 60%, in certain parts of India the mortality rate from VL is very

Chapter 72: Vaccines Against Leishmania Table 1 Burden of Disease in DALYs in WHO Regions, Estimates for 2002 Geographical regions

DALYs

Western Pacific Europe The Americas Middle East Southeast Asia Africa

50,000 6,000 44,000 48,000 1.358 million 383,000

Total

2.09 million

Abbreviation: DALYs, disability-adjusted life years.

high (13). The pattern of disease has changed dramatically in the past decade in South Western Europe where HIV infection has shifted what was a pediatric disease to one of adults. In different countries of Southern Europe 25% to 75% of all cases of VL emerge from people infected with the HIV. Moreover, 1.5% to 9.5% of all patients with acquired immunodeficiency syndrome (AIDS) suffer from newly acquired or reactivated VL (14) in different countries of Europe. Leishmaniasis is present in all continents, but is restricted to temperate climates (458 North to 358 South) perhaps because of survival and activity period of the vectors. More than two dozens sandfly species are vectors of leishmaniasis (Lutzomyia in the Americas and Phlebotomus in the rest of the world). Transmission by contaminated needles in drug abusers has been documented (15). Leishmaniases have diverse epidemiological characteristics and transmission occur intra- or para-domiciliary, or in the wild, with forests, deserts and mountains making reservoir or vector control difficult or impossible. The first-line drug (antimonials) developed almost a century ago require repeated injections (four weeks of daily injections for VL), are costly, often associated with side effects and are becoming ineffective in many endemic foci. Oral Miltefosine and injectable paromomycin have recently been registered against VL and are being used as monotherapy. These drugs have limitations (potential toxicity, and compliance) and if they continue to be used as single agents, they will soon become useless because of selection of resistance. A therapeutic vaccine combined with these drugs would prolong their usefulness in the field. Antigenic cross-reactivity among Leishmania is the rule rather than the exception, hence it is hoped that with a single vaccine it might be possible to protect against different Leishmania species. Some experimental and epidemiological data support this notion (16,17) but exception is also seen in experimental leishmaniasis (18). Several reviews have been published on vaccines against leishmaniasis (19–23). Here we emphasize those that have been in development through clinical trials. Although antigen selection studies have led to identification of many candidate molecules that are protective in animal models (see second-generation vaccines, below) only one has been taken into clinical development. This is primarily because the market for a leishmaniasis vaccine is conceived to be very limited and only of local importance within the endemic regions. Most research laboratories do not have the resources or the expertise required for preclinical and clinical development and the large pharmaceutical industries are not able to invest the resources required because of the small market. The best solution is to promote a vaccine producing facility of a developing country where leishmaniasis is of public health importance to good manufacturing practices (GMP) standards and seek local governments’ support for purchasing and

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distribution. Philanthropic support for transfer of technology and production under GMP conditions is needed as most donor agencies for medical research are not interested in development.

IMMUNOLOGY OF LEISHMANIASIS Acquired resistance to leishmaniasis is mediated by T cells. T cell–deficient mice rapidly succumb after infection with most species of Leishmania and adoptive transfer of normal T cells confers resistance to the animals. Moreover, as mentioned before, patients with AIDS are highly susceptible to leishmaniasis either as a result of concurrent infection or as a reactivation of older subclinical infection (4). Among the T cells, CD4þ are crucial for resistance against L. major while CD8þ T cells seem to participate more in the memory events of the immune response than as effector cells involved in parasite elimination (24–36). In experimental VL models, CD8þ T cells were shown to be required for late stage hepatic resolution (37) and for protection mediated by a subunit vaccine (38). These conclusions were reached from a series of experiments using mice genetically engineered to lack class I or class II MHC molecules, in mice lacking CD4 or CD8 cells, and in C1qa
/
mice. In the case of CL, effective protection has been largely attributed to the production of IL-12 and IFN-g, which mediate macrophage activation, nitric oxide production and parasite killing (39). Using multiparameter flow cytometry to assess the immune responses following immunization, Seder and colleagues recently demonstrated that the level of protection against L. major infection in mice is predicted by the frequency of CD4þ T cells simultaneously producing IFN-g, IL-2 and TNF (40). A clear-cut polarization of T helper cell responses is not evident in human leishmaniasis, which shows a mixed Th1 and Th2 immune response (41). The spectrum of susceptibility of different strains of mice to L. major infection has been extremely helpful for the understanding of genetic control of the disease and the mechanism of protection or susceptibility mediated by different subsets of CD4þ T cells (42–45). Hence, the outcome of L. major infection in mice is under a multi-gene control system (46). Most of these genes do not map in the MHC systems of either humans or mice (47,48). The resistant strains such as C3H, C57BL/6, CBA/J, or B10D2 normally develop a small lesion, which heals spontaneously within four to six weeks. While the BALB/c strain is susceptible to the extent that a few metacyclic parasites will cause a full blown lethal disease that is difficult to treat with first line drugs (antimonials). The BALB/c mice develop a progressive local lesion and a systemic, visceralized disease at later stages of the infection (49) with a pathology somewhat similar to human VL, including hepatosplenomegaly and lymphadenopathy (50). In addition, this outcome is directly related to the typical Th2 response observed in these animals (51). Mice of the resistant phenotype clearly develop a dominant Th1 phenotype of immune response to the parasite’s antigens and interference with this response will make them susceptible (52,53). Similarly, innate immunity, including natural killer cells, IL-1 alpha and myeloid differentiation factor 88 (MyD88) act as immunomodulators determining early resistance to infection (54). The skin is a site preconditioned for early parasite survival by virtue of a high frequency of steady state, natural CD25þFoxp3þ regulatory T (Treg) cells that function to suppress the generation of unneeded immune responses to infectious and noninfectious antigens to which the skin is regularly

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exposed. In murine models of infection, antigen-induced CD25+ Foxp3 interleukin (IL)-10þ Treg cells act during the effector phase of the immune response to control immunopathology and may also delay or prevent healing. Finally, following resolution of infection in healed mice, CD25þFoxp3þ Treg function in an IL-10-dependent manner to prevent sterile cure and establish a long-term state of functional immune privilege in the skin (55). Using a clinical isolate of L. major (NIH/Sd strain) that causes infected nonhealing lesions, an important role for Foxp3
Th1 cells that coproduce IL-10 and IFN-g in the response against these Leishmania parasites was demonstrated (56). Interestingly, Treg cells were shown to be crucial for the inhibition of a TH2-cell response that would normally develop in their absence. This Th1 cell response would, in turn, switch off the more efficacious Th1-cell response. However, in a different model of L. major infection, in which the mice can be clinically cured by a Th1-cell response (the Friedlin strain), the production of IL-10 by Treg cells regulates the immune response so that cure and eradication of the parasite requires neutralization of IL-10 (57). Nevertheless, these studies indicate that the relative levels of IFN-g and IL-10 produced by Th1 cells may influence the balance between clearance of infection and persistent infection in the response against certain pathogens (58), and this thereby determines whether immunopathology or chronic infection ensues. In contrast to T cells, B cells are apparently important only during the early events involving the development of parasite specific immune response. B cells, the specific antibodies they produce, and complement are involved in host effector mechanisms against the parasites particularly as they act as endogenous adjuvants for vaccine-induced CD8þ T-cell responses (38). Nevertheless, when B cell–deficient mice of the resistant phenotype were infected with either L. mexicana or L. donovani, they did not develop disease (59–61). The role of Fc g receptor I and Fc g RIII-mediated uptake of L. major amastigotes by DC is also an important factor for Th1 development (62). Hence B cells may play a role in curtailing infection in cutaneous leishmaniasis—at least in some experimental models. In humans, circumstantial observations that show a good correlation between Th1 response and resistance are primarily applicable to CL. A predominance of IFN-g producing cells has generally been found in healing cutaneous lesions while in chronic cutaneous or mucosal lesions a mixture of type-1 and type-2 cytokines with striking abundance of IL-4 mRNA has been consistently found (63,64). In VL however, no association with increased IL-4 and active disease could be found. Both splenic IFN-g mRNA and IL-4 mRNA are elevated during active disease, and decline significantly after cure. This same pattern of cytokine profile production occurs after antigenic stimulation of peripheral blood monocytes (PBMC) of patients with active disease and after cure (65). However, a direct correlation between IL-10 production and active disease was reported in VL patients (66,67). In conclusion, the human studies similar to the murine observations, point to a preferential association of Th1 cytokines with resistance against cutaneous leishmaniasis. Therefore, the design of a vaccine against CL should involve immunization protocols that generate primarily IFN-g and little or no IL-4 responses to the leishmanial vaccine components. Recent studies on the generation and maintenance of central memory (CM) and effector memory (EM) CD4 þ T cells during leishmaniasis has provided some insight on the

design of effective vaccination strategies against Leishmania (68,69). In the murine model of disease, it has been suggested that the continuous presence of live parasites is required for maintaining EM CD4þ T cells, but might not be essential for the maintenance of CM CD4þ T cells (70,71) Thus, the efficacy of killed or subunit vaccines might be greatly enhanced by using adjuvants that favor the generation of CM CD4þ T cells. However, the importance of persistent infection for maintaining an effective and durable protective response is controversial (72,73). Since vaccines need to generate immunological memory, a better understanding of the formation and maintenance of CM and EM CD4þ T cells in both animal models and human disease will be critical for their development.

ANIMAL MODELS FOR VACCINE STUDIES Many Leishmania species infect mice, hamsters, and nonhuman primates. In addition, the natural hosts of some parasites, that is, Psammomys (the wild rodent for L. major) and dogs (for L. infantum) have been used as laboratory animals. VL vaccination studies have been hampered by the lack of a suitable animal model of disease. Several clinical symptoms and pathogenic features of infection in both dog and hamster models are similar to the human disease. The canine model is particularly useful in evaluating vaccine candidates since successful vaccination of dogs is thought, at least to some extent, to control the spread of disease to humans in endemic areas where the dog is the reservoir of infection (74). However, both models that use outbred animals also suffer from lack of immunological reagents needed for the dissection of correlates of protective immunity. The mouse model of VL has been the most widely used system. It has the advantage that there are many different knockout mice with specific lesions in the immune system and there are good immunological reagents. However, it does not fully reproduce the disease observed in humans. The Th1 and Th2 polarization has not been observed for L. donovani or L. infantum and often the mice have to be injected intravenously with large numbers of amastigotes to achieve visceral disease (75). These models have been very important in studying the parasite biology, natural history and particularly the immunology of leishmaniasis as mentioned above. However, none of these animal models have been validated for vaccine studies for human diseases. Although a few investigators have used the resistant strains, the BALB/c and L. major infection has been most extensively used for vaccine studies. It is thought that if a vaccine can protect BALB/c mice, then it should also protect humans. The validity of the BALB/c model has been supported by studies showing that crude (20) or defined (19) vaccine candidates that protect the BALB also protect rhesus monkeys. Laboratory animals are usually challenged by cultured promastigotes without the components of sandflies, which have profound impact on the fate of the infection (76). Sometimes amastigotes are injected IV, when promastigote injection intradermally does not produce rapid or uniform infection, that is, in dogs, or infection with L. donovani in mice and hamsters. Skin-associated immune responses are crucial for protection against all forms of leishmaniasis as the port of entry is skin. Hence, a vaccine may protect against IV injection of amastigotes but have no impact against natural disease (77), or it may be that a vaccine would not be protective in an animal model (78), but it remains to be seen if it would protect humans. To promote infection, promastigotes isolated from infected sandflies just prior to injection of dogs (79) or exudates of salivary

Chapter 72: Vaccines Against Leishmania

glands of sandflies mixed with cultured promastigotes have been used in mice (76) and monkeys (80). Belkaid et al. (81) have developed a model by which infected sandflies deliver the parasite to the ears of mice. This is the most natural mode of delivering the challenge organism; however, it is not easily quantifiable. In most models of experimental leishmaniasis, parasite load is measured either by excising parts of an organ (foot, site of infection) or taking a biopsy or a smear imprint from liver, spleen or bone marrow to count the parasites either by direct counting or limited dilution techniques. These are inaccurate and labor intensive. More recently by introducing a gene of an enzyme (firefly luciferase, incorporated in the genome of L. major) (82), the real-time parasite load can be quantified in vivo. This is an accurate, simple and very useful tool for parasite enumeration, although probably not as sensitive as limited dilution. However, it allows the progression of infection or its regression as a result of immunological or drug intervention in real time without sacrificing the animal.

TARGET ANTIGENS FOR VACCINE DEVELOPMENT Leishmaniasis is a parasitic diseases that has a high potential of being prevented or treated by effective vaccines. Not only does recovery from a primary infection often result in resistance to subsequent infections, but the life cycle of the parasite is relatively simple. Leishmania exist in two principal forms: promastigote, the flagellated form in the invertebrate host that can be grown in cell free tissue culture media and amastigote, the round intracellular form in the vertebrate’s macrophages. There is some differential antigen expression in these two forms and most vaccine candidates are selected to be present at least in the amastigotes, though their presence in promastigotes as well may be an added advantage. The promastigotes also undergo antigenic changes in the process of maturation from procyclic (noninfective attached to the wall of the midgut of sandflies) to infective metacyclic form (released to the foregut for delivery during blood meal). This maturation also occurs in culture for most Leishmania. These antigens, essentially lipophosphoglycans of the promastigote surface are considered as possible antigens for transmission blocking vaccines as are components of sandfly guts (83–85). Specific antibodies to these antigens transferred to the sandfly during the blood meal could in principle prevent normal maturation of the parasite within the vector. Another intriguing target for vaccine development against leishmaniasis is the sandfly saliva. Recent observations in mouse models point to a protective effect of the phlebotomine’s saliva components against challenge with L. major. The mechanism of this protection has not yet been completely elucidated. However, it has been suggested that immunization of mice with the saliva of P. papatasi induces a strong delayedtype hypersensitivity (DTH) to the saliva components. When parasites are delivered with salivary gland excretions during a blood meal a local DTH reaction is induced which could mediate parasites killing (86,87). That a DTH to unrelated antigens at the site of infection could enhance healing was shown long ago (88). Inclusion of sandfly components that may induce hypersensitivity in a vaccine given to normal individuals who are exposed to bites of sandflies (mostly uninfected) may not be without risks. Further studies are needed to validate this approach.

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VACCINES IN USE There is no current prophylactic vaccine available for general use against leishmaniasis. ‘‘Leishmanization’’ an ancient preventive practice is still used in high-risk population in Uzbekistan (89,90). Leishmanization is the inoculation of a live virulent L. major at a selected site of the body to produce a self-healing lesion to induce a lifelong immunity to cutaneous leishmaniasis Presently a mixture of dead and live L. major is given at a covered part of the body which usually produces a lesion of 1 to 2 cm in diameter that lasts three to four months and heals spontaneously in a high endemic focus of Uzbekistan, mostly to school age children and migrants to the area. The lesion will induce a protective response in more than 95% of recipients against natural infection, which is usually presented with multiple lesions on the face and other exposed parts of the body (Fig. 1). Resistance to reinfection following recovery from CL was known to be very high when the natural history of the disease was described more than 500 years ago (91). In its initial practice, pus from an active lesion was used for inoculation. After the parasite could be grown in vitro, axenically cultured L. major was used for inoculation (92,93). The Israeli group also used leishmanization in the 1960s and 1970s on over 5000 high-risk individuals (94). It was noted that the ‘‘take’’ rate of the vaccine dropped precipitously (85–15%) over the years using the same organism, which was repeatedly subcultured (95). To overcome this, a simple method was developed to produce stabilates by freezing the parasite, which preserved its virulence for long periods (96). Nevertheless the program was discontinued because of unwanted side effects, including allergic response in pre-exposed individuals, long duration of active lesion and lack of immunity in the ‘‘nontake’’ individuals. Leishmanization was practiced in large scale primarily in soldiers as the last resort in Iran during the IranIraq war in the 1980s (61), following a trial in civilians (62), which showed over 98% efficacy. The leishmanization program was stopped after the war mainly due to the prolonged duration of lesions and appearance of a few nonhealing cases that are very difficult to treat.

LEISHMANIZATION AS A LIVE CHALLENGE INFECTION TO EVALUATE EXPERIMENTAL VACCINES A major time and cost strain for development of a vaccine is the conduct of pivotal field efficacy (phase 3) trials with sufficient power (large sample size) to obtain statistically significant results for registration. Live challenge systems are very powerful tools in vaccine development. Hence prior to embarking on costly phase 3 trials of a candidate vaccine, live challenge can provide very good information on its efficacy in humans. This is particularly important when true surrogate markers of protection are not fully identified—as is the case with leishmaniasis. Live challenge studies have been done for malaria (97) and cholera (98) and are developed under controlled conditions in HIV-free areas for evaluating vaccines against leishmaniasis. In two trials to evaluate reproducibility of leishmanization using the same stabilates after about three years of storage, remarkable similarities were observed with respect to duration and severity of lesions produced (99). Because of small sample size and controlled infection, live challenge is a very useful tool for detailed immunological studies in search of surrogate markers of immunity (100). In addition, unlike other diseases mentioned

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Figure 1 Lesions induced by leishmanization and by natural infection.

above, everyone entering in a leishmaniasis vaccine trial that includes leishmanization as live challenge will be protected against the disease, either by the experimental vaccine or by leishmanization. Considering the benign nature of the lesions developed by leishmanization and the site of the lesion, in usually covered part of the body (upper deltoid), compared with unsightly lesions on the face by natural infections (Fig. 1), there is a high willingness in certain hyperendemic areas to participate in a vaccine trial with leishmanization.

Live Leishmania Vaccines Live Leishmania vaccines have general drawbacks and none have reached the stage of preclinical development. Since live Leishmania cannot be lyophilized to maintain viability and they transform in vitro with differential expression of genes, standardization and delivery to many leishmaniasis endemic foci would be a major obstacle. Leishmania can be kept viable frozen in liquid nitrogen, but delivery to the population at risk would require much infrastructure beyond the means of many endemic countries. In addition, with the danger of present expansion of HIV infection, live Leishmania vaccines are unlikely to find wide acceptance, since nonpathogenic parasites can coinfect and have been isolated from HIV-infected patients.

Using wild-type virulent L. major for leishmanization (LZ), a lesion must develop before protection is induced. However, there are examples of protective vaccination in mice with genetically modified parasites (either by mutagenesis (101) or by genetic modification (102) that do not produce the pathology, yet induce protection against wild-type parasites under certain conditions. Another approach for genetic modification is to add external gene(s) ‘‘suicidal cassettes’’ (103) to render the parasite more responsive to drugs. In all these models, the idea is to induce protection but not the pathology associated with LZ. Knockout Parasites The first construct generated by gene targeting, was a dihydrofolate reductase-thymidylate synthase (DHFR-TS) L. major knockout (102), Injected mice showed significant protection (short term) against challenge with wild type (104). Although the knockout parasite showed a type-1 cytokine response by human cells in vitro, further studies in monkeys were disappointing (105) hence the DHFR-TS knockout construct has not been further developed as a vaccine. Using a similar technique of homologous recombination with L. donovani, the biopterin transporter (BT1) was inactivated. The BT1 knockout construct had much-reduced

Chapter 72: Vaccines Against Leishmania

virulence but elicited an immune response leading to a high level of resistance against challenge by the wild-type parasite (106). Several cysteine proteinases (CPs) of L. mexicana knockouts were constructed lacking CPa, CPb or both (107,108). These constructs had reduced pathogenicity and induced partial protection in BALB/c mice against challenge with the wild-type L. mexicana (109). Suicidal Cassettes A double drug sensitive strain of L. major was constructed (110) by introducing HSV-1 thymidine kinase gene (to confers increased sensitivity to ganciclovir) and a Saccharomyces cerevisiae cytosine deaminase gene (for sensitivity to 5fluorocutosine). Progressively growing lesions in BALB/c mice, with this construct, were completely cured by two weeks of treatment with either drug alone or in combination. Treated animals showed no signs of recurrence of infection for at least four months when the experiments were terminated (111). None of these constructs has reached clinical development yet, however the approach provides possibilities for induction of protection with a self-limiting infection, possibly without any pathology.

FIRST-GENERATION VACCINES (KILLED LEISHMANIA WITH OR WITHOUT ADJUVANTS) Whole killed parasites if given by appropriate route with adjuvant can protect many experimental animals and hence have been used as a golden standard to evaluate different vaccine candidates. Since most Leishmania species can easily be grown in cell free cultures, killed parasite has been tried as a vaccine much in the same way as early bacterial vaccines. The history of vaccine trials using killed Leishmania goes back to 1920s and 1930s first for immunotherapy and then in 1940s for prophylaxis [reviewed by Genaro et al. (112)]. In the 1970s and 1980s, Mayrink and his group in Brazil following the earlier studies of Pessoa (113) and Convit and colleagues using BCG as adjuvant in Venezuela initiated vaccine trials with killed Leishmania in prophylactic as well as therapeutic trials (see below). The outcome of all these trials have revealed that whole killed parasite alone or mixed with BCG (as adjuvant) are safe but only weakly immunogenic, hence not useful as prophylactic vaccine. However, they may be very good as an adjunct to chemotherapy either to reduce the dose of drugs, duration of treatment or both (see therapeutic vaccines, below).

New World, Multiple Strains (Mayrink’s Vaccine) The initial vaccine of Mayrink consisted of 5 different Leishmania and several trials were conducted (114–116). Three injections were given IM one week apart to volunteers with a negative leishmanin skin test (LST) (also called a Montenegro skin test). The antigen is a low concentration (5–10 mg) phenol killed parasite. Vaccination induced LST conversion (>5 mm induration after 48–72 hours) in 35% to 70% of volunteers in different trials ranging from 480 to 2500 volunteers in each trial. The vaccine was well tolerated; acute adverse reactions were rare (mild pain) and long-time follow-up showed no untoward responses, including presence of autoantibodies. Collectively the trials showed the safety of this approach and revealed that skin test conversion as a result of vaccination is a useful tool in

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field studies to monitor responsiveness of the population. Antunes et al. (114) demonstrated that LST converters have a lower incidence of disease, which has been repeatedly seen in subsequent trials (see ALM trials). A three-species autoclavekilled vaccine was produced in the laboratory of Armijos et al. (117) and the safety, immunogenicity, and efficacy of two injections against cutaneous leishmaniasis was tested in rural Ecuadorian children in a randomized, BCG-controlled, doubleblinded study. Live BCG was used as adjuvant. Within the oneyear follow-up, the incidence of CL was significantly reduced in the vaccine group compared with the control group (2.1% vs. 7.6%, p < 0.003). The protective efficacy of the vaccine was 72.9% (95% confidence interval ¼ 36.1–88.5%). This is the only trial in which a significant difference was observed between killed Leishmania þ BCG versus BCG alone.

New World (Single Strain) Brazil A single-strain L. amazonensis vaccine was produced by Biobras, after careful comparison of different strains (112). This vaccine was tested in a dose escalating trial for safety and skin test conversion (118) and further trials were conducted to analyze the immune responses (119–121). The vaccine induced primarily a Th-1 type response with demonstrable IFN-g but mostly from CD8þ cells—a pattern associated with the healing process in mice (122) and humans (123). This vaccine was effective (96% cure) when added to low-dose antimonial for treatment of CL in Brazil (see Therapeutic Vaccines below). Colombia Mayrink’s vaccine was formulated by Biobras at higher concentrations for use in combination with BCG, which is given ID. This formulation was compared for safety and immunogenicity with the IM formulation in a double-blind, randomized placebo-controlled trial in Medellin, Colombia (124). Because of side effects of BCG (active lesion for about three weeks, followed by scar formation) volunteers refused to receive the third injection; hence a comparative study could not be completed. Nevertheless, the three injections of Mayrink’s vaccine (killed parasite without BCG) were shown to be well accepted with minor side effects. There were 86% and 90% LST conversion on day 80 post vaccination and a year later, respectively. No antibody production to the vaccine antigens was seen and the cytokine pattern was that of a Th-1 response. On the basis of these results, a double-blind randomized, placebo-controlled efficacy trial was conducted in Colombia on a total of 2597 healthy volunteers with negative LST. The participants were selected from rural Colombian soldiers who were going to patrol endemic areas. Safety and efficacy of the vaccine were determined by comparing local and systemic adverse reactions after each dose and the incidence of parasitologically confirmed CL. The vaccine was shown to be safe but offered no protection against CL caused by L. panamensis. Unfortunately, there was no LST performed after vaccination to evaluate the immunogenicity of the vaccine and determine if the converted LST subpopulation had a lower incidence of disease as was reported in previous trials (125). Venezuela Convit and colleagues were the first to use BCG as an adjuvant and autoclaved Leishmania as the immunogen for immunoprophylaxis as well as immunotherapy (126,127). Without the addition of antimonials, three injections of the vaccine

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(autoclaved L. mexicana þ BCG) given a month apart can cure about 90% of CL patients (slightly below drug treatment), but at a much lower cost and with no serious side effects. In contrast drug treatment (60 injections) produced 18% serious (cardiopathy, renal toxicity) and 16% moderate adverse reactions. Although drug treatment reached over 90% cure in a shorter time, immunotherapy (three injections) is far more applicable and cost-effective than drug treatment. BCG treatment alone cured about 40% during the same period. Historical controls without treatment usually take much longer to heal. This treatment is now being given first and if patients do not respond to three injections, they will be treated with chemotherapy (128). Recently, Convit and colleagues have modified the preparation by using pasteurization instead of autoclave and have shown efficacy in treating mucosal and early lesions of diffuse leishmaniasis (129).

Old World (Killed Leishmania major þ BCG) Following establishment of a seed bank from the L. major, which was used in the mass leishmanization program (see above), the Razi Institute in Iran produced different formulations of killed parasite, which were tested in randomized double-blind and controlled trials with a single and multiple injections for safety, dose finding and immunogenicity (130). Finally, autoclaved L. major (ALM) produced similarly to Convit’s method was chosen for efficacy trials at a dose of 1 mg/injection. This preparation has been used for all further trials of ALM þ BCG in single or multiple injections against homologous or heterologous parasites (131–133). All studies were double-blind, randomised and BCG controlled. BCG rather than a placebo was used to assure blindness even though BCG clearly converts some individuals (5–15%) from LST
to LSTþ (>5 mm), has a nonspecific immunopotentiation and antigenic cross-reactivity with Leishmania. The results of three efficacy trials with single injection of ALM-BCG against zoonotic CL (132), anthroponotic CL (133) and two injections against VL (131) were conclusive in that the vaccine was safe and induced LST conversion in certain individuals (17–36% of target population), In contrast to the trial of Armijos (117), in none of these trials with ALM þ BCG versus BCG, either with single or up to three injections, was there a significant difference between the two groups. However individually and collectively these trials produced interesting findings as indicated below:

1.

2.

3.

In all the trials the vaccine produced more skin test conversion than BCG alone as expected, but BCG alone also produced LST conversion in 3.2% to 7.9% of individuals when tested 40 to 80 days post injection and as high as in 35% after a year. In all the trials the LST converted individuals had a reduced incidence of disease. This was significant for the homologous trial and the trial with two injections against VL but although the same trend was seen the difference was not significant (p < 0.6). These findings confirm the earlier studies with Mayrink’s vaccine (114). The rate of skin test conversion in the volunteers living in the nonendemic regions was significantly higher than that in the endemic regions. This indicates that immunogenicity studies must be conducted in the endemic foci prior to deciding on the dose for formulation in efficacy trials. The reason for this is not clear, however the LST-negative individuals living in an endemic focus may be genetically

4.

5. 6.

‘‘nonresponders,’’ whereas those in the nonendemic focus would be mixed. BCG alone induces LST conversion and may protect a small proportion of volunteers for a short period of time. BCG is known to have cross-reactive antigens with Leishmania and to stimulate the cellular immune response nonspecifically for a limited period of time. Hence the use of BCG as control in these trials may have obliterated the true protective value of the vaccine. The vaccine is not sufficiently immunogenic as presented and stronger adjuvants should be used to enhance its immunogenicity. BCG is required in the vaccine to induce LST conversion in the first injection as well as in the booster injections (134).

New Formulation of ALM þ BCG To increase immunogenicity of ALM, Razi Serum and Vaccine Research Institute, Iran, developed a new formulation by adding alum to the vaccine. Single injections of Alum-ALM plus IL-12 or BCG were shown to protect against CL, caused by L. amazonensis (135) and against a lethal challenge with L. donovani in nonhuman primates (136). The dose escalating safety and immunogenicity trials in Sudan showed that alum– ALM þ BCG was safe and far more immunogenic that ALM þ BCG (137). This formulation is now in a clinical trial for efficacy against leishmanization in Iran. A phase 2 study, further established the safety and immunogenicity of this formulation and there was an indication that it may be protective against VL in Sudan (138). A total of 544 participants were randomized and injected either with a single dose of the vaccine or vaccine diluent as placebo. At the end of two-year follow-up, there were four cases in the placebo arm and none in the vaccinated group. Following a very encouraging hospital-based preliminary but definitive trial using alum–ALM þ BCG as an adjunct to antimony treatment in persistent post-kala-azar dermal leishmaniasis (PKDL) cases in Sudan, a field trial is being planned (see Therapeutic Vaccine below).

SECOND-GENERATION VACCINES Anti-Leishmania Subunit Vaccine Composed of Recombinant Proteins Several investigators, have over the past decade, searched for genes encoding leishmanial proteins that could induce protection against cutaneous and VL in several experimental models of the disease. Table 2 contains many of the recombinant proteins that have been described and obtained using a variety of cloning strategies (139–153). Studies of recombinant protein vaccines in mice demonstrated that antigens such as the proteins GP63, p36/LACK, A-2, gp46/M-2/parasite surface antigen 2 (PSA-2), P0, LCR1, HASPB1, ORFF, Q protein, and KMP11 induced immune responses, but short-lived protection against Leishmania infection (148,154–158) (Table 2). Other studies have identified several antigens, including lmd29 and 584C, that reproducibly exacerbated leishmania disease (222). The GPI-anchored membrane protein PSA-2, which consists of leucine-rich repeat motifs (LRRs) belongs to a gene family present in all Leishmania species except L. braziliensis (223). There are three distinct L. major PSA-2 polypeptides expressed on the promastigote surface, but only one on amastigotes (224). Immunization with the three polypeptides of

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Table 2 Second-Generation Vaccines Second-Generation Vaccines Against Leishmaniasis Disease targeted/outcome of vaccination

Antigen

Vaccine

Animal model

LPG

Native protein Native protein þ BCG Recombinant protein Native protein Protein expressed in BCG protein in Salmonella DNA Peptide-pulsed DC Protein-pulsed DC

Mouse Mouse, hamster Mouse Monkey Mouse Mouse Mouse Mouse Mouse Mouse

gp46

Native protein Protein expressed in vaccinia virus DNA

Mouse Mouse Mouse

CL/protection, CL/VL/no protection CL/no protection CL/partial protection CL/protection CL/protection CL/protection CL/protection CL/partial protection CL/variable protection CL/protection CL/protection CL/protection CL/variable protection

p36/LACK

Recombinant protein þ IL-12 DNA/recombinant protein expressed in vaccinia virus DNA/recombinant protein expressed in Salmonella or Listeria DNA Peptide-pulsed DC Recombinant protein

Mouse Mouse, dog Mouse Mouse Mouse

CL/protection CL/VL/protection CL/protection/partial protection CL/no protection/protection CL/protection

Dog Monkey Mouse Mouse Mouse

186 187 156,188

Dog Hamster Mouse Mouse Mouse Mouse Mouse Mouse Mouse Dog Hamster Mouse Mouse Mouse Mouse Mouse Mouse Mouse Dog

VL/protection CL/partial protection CL/partial protection CL/partial protection CL/partial protection No protection VL/protection VL/protection CL/protection VL/partial protection VL/partial protection VL/protection VL/protection CL/partial protection VL/protection VL/protection VL/partial protection CL/protection VL/partial protection VL/protection VL/protection CL/protection CL/protection CL/protection VL/protection

Mouse Mouse Mouse Mouse Mouse; monkey Mouse; monkey Mouse

CL/VL/protection CL/protection VL/protection VL/protection CL/protection CL/protection CL/protection

207 208 209 210 140,211,212 140,153 213,214

Dog

VL/protective immunity

215

DNA

Mouse

CL/protection

216

Recombinant protein Recombinant protein Protein expressed in adenovirus

Mouse Dog Mouse

Recombinant protein

Mouse

CL/VL/protection VL/no protection VL/protection CL/protection VL/protection

19,217–221 78 186 40 210

gp63

H1 CPB CPA þ CPB CPA þ CPB KMP11 LCR1 A2

HASPB1 PapLe22 P8 ORFF

P4 PFR-2 Lip2a þ Lip2b þ P0 þ H2A FML/NH36 gp63 þ NH36 NH36 SMT LmSTI1 TSA LmSTI1 þ TSA LmSTI1 þ TSA þ LeIF LmSTI1 þ TSA þ LACK Leish-111f

SMT

Recombinant protein DNA DNA fusion/recombinant fusion protein DNA/ recombinant protein DNA Antigen-pulsed DC Recombinant protein Protein expressed in BCG DNA Recombinant protein Native antigen Recombinant protein Recombinant protein DNA Native protein Recombinant protein DNA/recombinant protein DNA Native protein DNA DNA Recombinant fusion protein DNA DNA DNA Recombinant Recombinant Recombinant DNA fusion Recombinant Recombinant

protein protein protein fusion protein proteins

Abbreviations: DC, dendritic cell; CL, cutaneous leishmaniasis; VL, visceral leishmaniasis.

References 159–162 163 148 150 141,164–172 173 155

144,145, 157,174 158,171,172, 175,176 147,177 178–182 183,184 171,185 172,176 155

156,188–190 191 189 154,192 155 193 194 143,195 143 196 152 186 197 196 198,199 200 201 202 203 204,205 206

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native promastigote PSA-2 protected mice against L. major (144), but vaccination with a recombinant E. coli–derived promastigote or amastigote protein showed lack of protective efficacy despite the ability to induce T h 1-type immune responses (157). Protective vaccination was also achieved against L. amazonensis (174). The kinetoplastid membrane protein 11 (KMP-11), a highly conserved surface membrane protein present in all members of the family Kinetoplastidae, is differentially expressed both in amastigote and promastigote forms of Leishmania is a vaccine candidate antigen (225). KMP-11, unlike gp63, for example, induced significant production of IFN-g from lymphocytes of patients cured of VL (226). A DNA vaccine encoding KMP-11 was evaluated for it is protective ability in genetically immunized hamsters. The protective effect was thought to be linked to the generation of functionally active IL-2-producing T cells along with specific anti-KMP-11 CTLlike response and other leishmanicidal effector mechanisms. Sterol 24-cmethyltransferase (SMT), was recently identified by serological screening using sera from L. infantum– infected hamsters (227). SMT is an enzyme involved in biosynthesis of ergosterol, which is a target molecule of leishmanicidal and fungicidal amphotericin B. The antigenicity, immunogenicity, and protective efficacy of SMT were recently evaluated (210). SMT formulated in MPL1-SE was found to be protective against L. infantum challenge perhaps through the induction of a Th1-type immune response with antigen-specific CD4þ and CD8 þ T cells. The rSMT plus MPL-SE vaccine induced SMT-specific T cells which were capable of producing multiple Th1-type cytokines (TNF, IL-2, and IFN-g) in response to Antigen recall. TNF, IL-2 and IFN-g are involved in protection against VL (228–230). As TNF synergizes with IFN-g in killing Leishmania parasites, induction of antigen-specific T cells capable of producing multiple cytokines upon antigen recall might be more beneficial for control of Leishmania infection than those producing a single cytokine. Antigens expressed in the amastigote stage may be the most important vaccine candidates since this form of the parasite is both the main inducer and the target of the immune response. The cathepsin L-like CPs are thought to be good vaccine candidates because of their high immunogenicity and important role in host-parasite interaction (231,232). Three classes of CPs have been identified: Type I (CPB), Type II (CPA) and Type III (CPC) (233). Immunization of mice with recombinant CP induced partial protection against L. major challenge (156) (Table 3). Recently, a hybrid fusion protein composed of CPA and CPB was used to immunize mice and dogs and partial protection against L. major infection was obtained (189,234). Few studies have been directed at the potential for a vaccine derived from one Leishmania species to provide crossprotection against another species. Initial results using sequential infections with distinct species have suggested complex cross-protection relationships. For example, immunization of mice with heat-killed L. donovani can induce protection against a subsequent infection with L. major (235). Few antigens, including LACK (146,172,176), dp72 (236), and P4 nuclease (203), have been tested for cross-protection in mice with varied success. In contrast to the strictly L. major species-specific protection with LACK, cross-species protective efficacy has been demonstrated for dp72. This protein, purified from L. donovani promastigotes was able to protect mice against L. major challenge (236). Cross-species protection is also a feature of the acidic ribosomal P0 protein from L. infantum that was able to

protect C57BL/6 from L. major infection. In this case, however, protection could not be induced in BALB/c mice, reinforcing the importance of host genetics (237). Soon after the great success in producing these recombinant antigens it became evident that a critical step in vaccine development was missing, that is, an acceptable adjuvant/delivery system capable of promoting the induction of an immune response biased toward Th1. Perhaps more critical than the choice of antigens for future vaccine development is the selection of an appropriate adjuvant or delivery system. Successful protection in mouse models has been achieved by vaccination with antigens delivered as DNA, or as proteins delivered with a variety of adjuvants. DNA encoding the leishmanial proteins LACK, LmSTI1, and TSA could effectively immunize susceptible BALB/c mice against L. major by inducing protective T-cell responses (213,238,239). However, DNA as a means to deliver prophylactic vaccines has lost its momentum, as studies in monkeys and man have yielded disappointing results. Similarly, cytokine adjuvants are not a practical alternative for vaccine development, but have nonetheless provided early proof of concept data illustrating the ability of crude protein preparations or of defined recombinant proteins to induce solid protection against disease in the mouse model. Both IFN-g and IL-12 have been used as adjuvants to induce antigen-specific protective Th1 responses (36,240). IL-12 injected subcutaneously with leishmanial soluble antigens (SLA), induces a strong anti-SLA Th1 response and no detectable Th2 response to this antigen. Importantly, this protocol of immunization confers excellent protection in BALB/c mice challenged with L. major (241). IL-12 has been successfully used as a Th1 adjuvant for a variety of antigens in both the murine and in the nonhuman primate models of several infectious diseases including leishmaniasis (135,140,241–243). However, in contrast with conventional adjuvants and with DNA immunization, it seems that the immunological memory to the immunizing antigen is not stimulated appropriately when IL-12 is used as adjuvant. Thus, vaccination of BALB/c mice with the leishmanial antigen LACK mixed with IL-12 as adjuvant resulted in short term protection against challenge with L. major. The two adjuvants approved for human use, alum and squalene, induce potent antibody responses but are poor inducers of antigen-specific Th1 responses. Protection against leishmania parasites appears to require the induction of IL-12 by antigen-presenting cells, which can be achieved via stimulation of toll-like receptor 4 (TLR-4), TLR-9, or TLR 7, or TLR 7/8. One recent study has reported that protection against leishmania is dependent on TLR-4 (244,245). Thus, monophosphoryl lipid A (MPL) (or related molecules with similar properties) is a logical choice as a vaccine adjuvant, as it stimulates TLR-4 and is a component of several experimental and two approved vaccines (Fendrix hepatitis B vaccine and cervarix, HPV vaccine, GSK). Building on the experience with MPL, a new synthetic adjuvant molecule, glucopyranosyl lipid A, or GLA, has been developed for next-generation Leishmania Vaccines. CpG ODN alone has been shown to induce a state of partial resistance in BALB/c mice for up to five weeks against challenge with L. major (246). If the CpG ODN is injected in conjunction with SLA significant protection is obtained in these animals that is maintained for as long as six months. In these experiments, the immunostimulatory properties of the CpG ODN were associated with production of IL-12 and the emergence of strong Th1 response to SLA (247,248). However further studies are required to optimize CpG ODN as an adjuvant for humans.

Chapter 72: Vaccines Against Leishmania

VACCINE DEVELOPMENT WITH THE RECOMBINANT ANTIGENS TSA, LmSTI1, AND LeIF On the basis of protection seen in mice and nonhuman primates (140,153,211,249) we have selected three leishmanial antigens, TSA, LmSTI1 and LeIF, to be included as a single protein in a recombinant vaccine. This vaccine, Leish-111f, will be developed both for prophylaxis as well as therapy for different forms of leishmaniasis.

TSA A novel protein of L. major with sequence homology to eukaryotic thiol-specific-antioxidant (TSA), it was discovered in experiments performed to characterize the immune responses elicited by L. major promastigote culture filtrate proteins (CFP). To identify immunogenic components of the promastigote CFP, serum samples from CFP vaccinated BALB/c mice, prior to challenge with L. major, were used to screen an L. major cDNA expression library. Southern blot hybridization analyses indicate that there are multiple copies of the TSA gene in all species of Leishmania that were analyzed (L. tropica, L. donovani, L. infantum, L. chagasi, L. amazonensis, L. braziliensis, L. guyanensis). Northern blot analyses indicated that the TSA gene is constitutively expressed in L. major promastigotes and amastigotes. Immunization of BALB/c mice with recombinant TSA protein resulted in the development of strong cellular immune responses and conferred protective immune responses against infection with L. major when the protein was combined with IL-12 (140,153).

LmSTI1 Screening of an L. major amastigote cDNA library with sera from L. major infected BALB/c mice identified one clone with strong homology with eucaryotic stress-inducible protein 1, designated as LmSTI1. LmSTI1 contains six copies of the tetratricopeptide consensus motif that is common to stress-inducible proteins. Recombinant LmSTI1 protein plus IL-12 elicited a mixed cellular response that was skewed toward a Th1 phenotype and protected susceptible BALB/c mice (140,211).

LeIF Leishmania Elongation and Initiation Factor (LeIF) was identified by expression cloning using sera from a patient with ML to screen a L. braziliensis genomic library. Immunoreactive antigens were purified and analyzed in patient T-cell assays for the ability to stimulate proliferative responses and preferential Th1 cytokine production. Several cDNAs were identified, one of which was LeIF, a L. braziliensis homologue of the eucaryotic initiation factor 4A, selected because this unique molecule has two important properties: (1) it is a powerful stimulator of the innate immune system for the production of IL-12, IL-18, and IFN-g, and therefore a Th1 inducer; and (2) because of its immunotherapeutic properties in mice infected with L. major (151,249–251). Subsequent to the studies in which we showed that these individual antigens are protective against experimental leishmaniasis in both mice and monkeys, the three proteins LmSTI1, TSA, and LeIF were engineered as a single polyprotein (tri-fusion or Leish-111f) in tandem (217). This polyprotein (Leish-111f) was then tested in protection experiments in mice challenged with L. major (218). In addition, the adjuvant MPL-SE1 was employed instead of IL-12. This adjuvant, a monophosphory lipid A derived from Salmonella minnesota

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plus the emulsifier Squalene has been proven to be an excellent modulator of Th1 responses (252). More importantly, this adjuvant is suitable for human use. BALB/c mice immunized with Leish-111f plus MPL-SE mounted a strong Th1 response to epitopes of the three individual proteins and more importantly they were totally protected against challenge with high dose of virulent L. major (106 metacyclic forms) (19,218). In subsequent studies, we evaluated this candidate in animal models of VL and used T and B cell ELISPOTs and flow cytometry, to examine the mechanism of the protective immune response against L. infantum (219). The results were the basis for selecting this construct for clinical development. Hence Leish-111f has now been produced under conditions of GMP at large scale and has undergone phase 1 safety and immunogenicity studies in the United States, Brazil, Peru, Colombia and India. Phase 1 and 2 studies are ongoing in Sudan and Peru, respectively. Transfer of technology and local production in a leishmaniasis endemic country will follow as soon as safety and protective efficacy are confirmed in clinical trials. A next-generation antigen combination, based on steroly methyl transferase (SMT) has shown excellent protection in animal models of VL and is currently being developed for clinical trials.

THERAPEUTIC VACCINES The effector mechanisms for prevention of leishmaniasis mediated by CD4, Th1 is different from those responsible for cure, requiring CD8 cytotoxic cells. Hence a vaccine that may not be efficacious for prophylaxis might be useful as a therapeutic vaccine. Indeed the first-generation vaccines, which have not shown efficacy for prophylaxis, show significant activity for therapy either alone or in combination with antimonials for either reducing the required dose of antimony or treatment of persistence and drug resistant leishmaniasis. Convit and his colleagues pioneered immunotherapy in 1980s using autoclaved L. mexicana þ BCG (126,127,130) and presently immunotherapy is being used in Venezuela. Those who do not respond to immunotherapy will receive chemotherapy. In Brazil killed L. amazonensis without adjuvant has been used for therapy on cases refractory to drug or immunosuppressed (112,253). More recently, a double-blind trial was conducted to see if the immunotherapy could be used with a low dose of antimonial (8 mg Sb/kg/day) instead of the standard (16 mg Sb/kg/day). Reduction of antimony dose is important for eliminating side effects of the drug and reducing cost. On the basis of the highly significant results (94% cure in combined therapy versus 8% in low-dose control) this vaccine was registered in Brazil as an adjunct to low-dose antimony treatment but not as a prophylactic vaccine (254). Alum–ALM þ BCG in combination with full doses of antimonial (20 mgSb/kg/day) was effective for the treatment of persistent PKDL (a very difficult condition to treat and believed to be the reservoir of VL) in a hospital-based, double-blind, randomized, antimony-controlled trial in Sudan (255). A combination of 4 recombinant antigens of Leishmania, plus GM-CSF with or without antimony was used for treatment of patients with advanced, refractory ML in Brazil (256,257). More recently, Leish-111f administered with MPl-SE as adjuvant has proven to be safe, and has accelerated clinical cure of CL and ML (personal communication, Dr. F. Piazza). Other trials with this candidate vaccine are in progress, and studies to further improve efficacy using alternative antigens and/or adjuvants are planned.

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CONCLUSION During the past decade, several well-designed, double-blind, randomized trials have been conducted using various preparations of killed Leishmania (whole parasite) with or without BCG as adjuvant. With the exception of one report from Ecuador (72% protection), little or no efficacy was seen. Lower incidence of disease was seen in those who responded by converting from negative to a positive DTH reaction (15–35% in different trials with different doses). Alum has been added to increase immunogenicity and this formulation has been shown in preliminary trials to induce skin conversion in all recipients. It remains to be seen if the new formulation can induce significant protection against leishmaniasis. It should be noted however that the first-generation vaccines are crude antigens and it is difficult if not impossible to standardize them. The BCG used as adjuvant, although the most widely utilized vaccine in the world, is not standardized and various strains have different activities. Hence, even if the first-generation vaccines show efficacy, there will still be a need to develop a well-defined, safe, efficacious and standardized vaccine. The alternative of developing a second-generation subunit vaccine composed of recombinant antigens is now moving from the laboratory bench to clinical trials. Over the past five years several Leishmania recombinant proteins have been tested as vaccine candidates in mice and more recently in rhesus monkeys. A mixture of three leishmanial antigens, TSA, LmSTI1, and LeIF, engineered in tandem as a polyprotein named Leish-111f, mixed with the adjuvant MPL-SE (Corixa Corporation/GlaxoSmithKline Biologicals, Seattle, WA) consistently induced protection in animals challenged with several Leishmania species. In view of these promising results, the first second-generation vaccine (Leish-111f plus MPL-SE) has been formulated under GMP and is undergoing clinical testing. This has become possible because of a generous grant from the Bill & Melinda Gates Foundation to the Infectious Disease Research Institute and the collaboration with Corixa Corp., Seattle. The vaccine will be tested both for its prophylactic as well as therapeutic efficacy. One condition of the grant is assurance of affordability of the vaccine, should it be shown to be efficacious. To this end, every effort is being made to transfer the technology of GMP production to suitable vaccine manufacturing facilities in one of the leishmaniasis afflicted countries to seek local government support for reduction of cost and sustainability, should the vaccine prove to be efficacious. Of the major human infectious diseases for which vaccines do not currently exist, leishmaniasis represents an opportunity for success. The induction of protective immunity appears to be exclusively T cell mediated, with a strong dependence on type I cytokines. Intensive work during the last two decades, made possible by molecular techniques, have completely changed the prospects for the development of a safe and effective vaccine for leishmaniasis. Of equal importance is the recent development of potent T-cell adjuvants, which have been shown to be safe and effective in both animal models and in clinical studies. The employment of such adjuvants will expedite the development of a range of new vaccines not possible a decade ago. Furthermore, the demonstration that both whole parasite and defined antigen vaccines can be used to treat incurable drug resistant leishmaniasis not only provides strong support for the concept of a leishmaniasis vaccine, but indicate that demonstration of therapeutic efficacy may speed the development of candidate vaccines. Finally, leishmaniasis

vaccine efforts are providing important insights into approaches for development of safe and effective T-cell vaccines for a range of human diseases.

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234. Zadeh-Vakili A, Taheri T, Taslimi Y, et al. Immunization with the hybrid protein vaccine, consisting of Leishmania major cysteine proteinases Type I (CPB) and Type II (CPA), partially protects against leishmaniasis. Vaccine 2004; 22:1930–1940. 235. Bebars MA, el Serougi AO, Makled KM, et al. An experimental vaccine providing heterologous protection for Leishmania species in murine model. J Egypt Soc Parasitol 2000; 30:137–156. 236. Rachamim N, Jaffe CL. Pure protein from Leishmania donovani protects mice against both cutaneous and visceral leishmaniasis. J Immunol 1993; 150:2322–2331. 237. Iborra S, Carrion J, Anderson C, et al. Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice. Infect Immun 2005; 73:5842–5852. 238. Gurunathan S, Sacks DL, Brown DR, et al. Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. J Exp Med 1997; 186:1137–1147. 239. Gurunathan S, Wu CY, Freidag BL, et al. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol 2000; 12:442–447. 240. Ahuja SS, Mummidi S, Malech HL, et al. Human dendritic cell (DC)-based anti-infective therapy: engineering DCs to secrete functional IFN-gamma and IL-12. J Immunol 1998; 161:868–876. 241. Afonso LC, Scharton TM, Vieira LQ, et al. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science 1994; 263:235–237. 242. Biron CA, Gazzinelli RT. Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr Opin Immunol 1995; 7:485–496. 243. Trinchieri G. Function and clinical use of interleukin-12. Curr Opin Hematol 1997; 4:59–66. 244. Hawn TR, Ozinsky A, Underhill DM, et al. Leishmania major activates IL-1 alpha expression in macrophages through a MyD88-dependent pathway. Microbes Infect 2002; 4:763–771. 245. Kropf P, Freudenberg MA, Modolell M, et al. Toll-like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major. Infect Immun 2004; 72:1920–1928. 246. Zimmermann S, Egeter O, Hausmann S, et al. CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis. J Immunol 1998; 160:3627–3630. 247. Stacey KJ, Blackwell JM. Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major. Infect Immun 1999; 67:3719–3726. 248. Walker PS, Scharton-Kersten T, Krieg AM, et al. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFNgamma-dependent mechanisms. Proc Natl Acad Sci U S A 1999; 96:6970–6975. 249. Skeiky YA, Guderian JA, Benson DR, et al. A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Th1-type cytokine profile and to produce interleukin 12. J Exp Med 1995; 181:1527–1537. 250. Borges MM, Campos-Neto A, Sleath P, et al. Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein. Infect Immun 2001; 69:5270–5277. 251. Probst P, Skeiky YA, Steeves M, et al. A Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis factoralpha production and expression of B7-1 in human monocytederived antigen-presenting cells. Euro J Immunol 1997; 27: 2634–2642. 252. Ulrich JT, Myers KT. Monophosphoryl lipid A as an adjuvant. In: Powell MF, Newmann MJ, ed. Vaccine Design: the Subunit and Adjuvant Approach. New York: Plenum Press, 1995: 495–524. 253. Da Cruz AM, Filgueiras DV, Coutinho Z, et al. Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses

Chapter 72: Vaccines Against Leishmania and remission of lesions associated with antigen immunotherapy. Mem Inst Oswaldo Cruz 1999; 94:537–542. 254. Machado-Pinto J, Pinto J, da Costa CA, et al. Immunochemotherapy for cutaneous leishmaniasis: a controlled trial using killed leishmania (leishmania) amazonensis vaccine plus antimonial. Int J Dermatol 2002; 41:73–78. 255. Musa AM, Khalil E., Mahgoub FA, et al. Immunochemotherapy of persistent post-kala-azar dermal leishmaniasis: a novel

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73 Vaccines Against Schistosomiasis Alex Loukas Queensland Institute of Medical Research, Queensland, Australia

Jeffrey M. Bethony Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A.

Andre´ Capron Acade´mie des Sciences De´le´gation aux Relations Internationales, Paris, France

Donald P. McManus Queensland Institute of Medical Research, Queensland, Australia

INTRODUCTION Schistosomiasis, caused by trematode blood flukes of the genus Schistosoma, is a major helminth infection that, at the beginning of the 21st Century, still represents an important public health problem in many developing countries. As the second major parasitic disease in the world after malaria, schistosomiasis affects 200 million people, with 800 million at risk of infection. It is estimated that 20 million individuals suffer from severe consequences of this chronic and debilitating disease responsible directly or indirectly for at least 500,000 deaths per year (1). There are three major schistosome species responsible for most cases of human schistosomiasis. Schistosoma mansoni occurs in much of sub-Saharan Africa, northeast Brazil, Surinam, Venezuela, the Caribbean, lower and middle Egypt, and the Arabic peninsula. S. haematobium is present in much of sub-Saharan Africa, the Nile valley in Egypt and Sudan, the Maghreb, and the Arabian peninsula. S. japonicum is endemic along the central lakes and River Yangtze in China, Mindanao, Leyte, and some other islands in the Philippines, and small pockets in Indonesia. S. japonicum is unique among the human schistosomes because it also completes its life cycle in water buffaloes—the issue of reservoir hosts in the control of human schistosomiasis japonica will be discussed later in the chapter. Schistosome infection is characterized by the presence of adult worms in the portal and mesenteric veins of humans and various mammalian species, as part of a complex migratory cycle initiated by cutaneous penetration of infective larvae (cercariae) shed by infected fresh water snails (Fig. 1). The infective larvae transform into schistosomula in the skin of appropriate hosts and, over several weeks, develop into sexually mature, egg laying worms. The adult worms can survive for up to 15 years in the definitive host. Female worm fecundity is characterized by the deposition in mucosae and tissues (in particular liver) of millions of eggs that are responsible for the pathology and disease associated with schistosomiasis. In spite of remarkable chemotherapeutic progress and the existence of highly effective molecules such as the acylated

quinoline-pyrazine, praziquantel (PZQ), schistosomiasis is still spreading into new areas. After more than 20 years’ experience, it is generally agreed that chemotherapy, although the mainstay of current schistosomiasis control programs (2–5), does have some limitations. In particular, mass treatment does not prevent reinfection. This rapidly occurs in exposed populations in most endemic areas so that within a period of six to eight months following chemotherapy, the prevalence returns to its baseline level. Furthermore, efficient drug delivery can require a substantial infrastructure to regularly cover all parts of an endemic area. This can make chemotherapy an expensive and often impractical approach. Although there is not yet clear-cut evidence for the existence of PZQ-resistant schistosome strains, decreased susceptibility to the drug has been observed (1,6), and in view of renewed efforts to control schistosomiasis in high-burden areas, particularly in Africa, by large-scale use of PZQ (3), there is increasing concern about parasite resistance developing. In the case of S. japonicum, despite widespread use of PZQ, especially in China, there is no evidence of PZQ-resistance, but an additional challenge is that transmission control necessitates interventions targeting the animal reservoirs, particularly buffaloes (1,7,8) (Fig. 2). Furthermore, in situations of ongoing high transmission and interrupted chemotherapy campaigns, severe ‘‘rebound morbidity’’ in terms of hepatosplenic disease is now well documented for schistosomiasis, contributing to the disease burden (9,10). As a result, vaccine strategies represent an essential component for the future control of schistosomiasis as an adjunct to chemotherapy. Vaccination can be either targeted toward the prevention of infection or to the reduction of parasite fecundity. A reduction in worm numbers is the ‘‘gold standard’’ for antischistosome vaccine development but, as schistosome eggs are responsible for both pathology and transmission, a vaccine targeting a reduction in parasite fecundity and egg viability is relevant. This article considers aspects of antischistosome protective immunity that are important in the context of

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Figure 1 The life cycle of Schistosoma mansoni, S. japonicum and S. haematobium. Source: From http://www.dpd.cdc.gov/DPDx/HTML/ Schistosomiasis.htm.

Figure 2 Water buffaloes, a reservoir host for Schistosoma japonicum, in a marshland area of China.

vaccine development. The current status in the development of vaccines against the African (S. mansoni and S. haematobium) and Asian (S. japonicum) schistosomes is then discussed, as are new approaches that may improve on the efficacy of available vaccines and aid in the identification of new targets for immune attack. Recent, comprehensive reviews of the area are available (11–27).

THE IMMUNE RESPONSE TO SCHISTOSOMES Most chronic morbidity in schistosomiasis is not due to the adult worms but is related to the T-cell-dependent immune response

of the host directed against schistosome eggs trapped in tissues. In the case of intestinal forms of the disease (S. japonicum and S. mansoni), this anti-egg pathology takes place mainly in the liver and intestines, and the bladder in S. haematobium. The trapped eggs produce a range of molecules leading to a marked CD4þ T cell programmed granulomatous inflammation involving eosinophils, monocytes, and lymphocytes. Granulomas are also characterized by collagen deposition and, in the intestinal schistosomes, severe hepatic periportal fibrosis occurs. Much of the morbidity and mortality associated with this disease is directly attributable to the deposition of connective tissue elements in affected tissues. In mice, a predominantly T-helper-1 (Th1) reaction in the early stages of infection shifts to an egginduced T-helper-2 (Th2)-biased profile, and imbalances between these responses lead to severe lesions (28–33). A notable accomplishment in the past few years was the identification of interleukin-13 (IL-13) and the IL-13 receptor (IL-13R) complex as central regulators of disease progression in schistosomiasis (30,34,35). IL-13 is the major cytokine that is responsible for fibrosis in egg-induced granulomas. Mice in which IL13/IL-13R is absent or neutralized with antibodies do not develop severe hepatic fibrosis during schistosome infection, leading to prolonged survival. Similar regulatory control could be at the basis of fibrotic pathology in humans (36), although this has not yet been established. Much of our understanding of the mammalian immune response to schistosomes is based on the use of gene-disrupted (knockout) mice (30,33,35,37,38) and the immunization of mice, nonhuman primates, or other mammalian hosts with irradiated

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cercarial vaccines with or without a subsequent challenge infection with non-attenuated cercariae (39–45). The attenuated larvae fail to mature to adult worms and do not produce eggs so that any results obtained are not confounded by egg-induced liver pathology. In general, these studies have established that T-cell-mediated immunity is fundamental to acquired resistance to schistosomes in mice, and that the irradiated cercarial vaccine was highly protective. Much of this protection was shown to be mediated by activated macrophages, and suggested that a vaccine that induced macrophage-activating Th1 cytokines (interferon-g and IL-2) may be beneficial in preventing schistosomiasis. However, repeated vaccination with irradiated cercariae produced incremental increases in Th2-mediated (IL-4 and IL-5 predominance) protection, which was transferable to non-vaccinated animals. In addition to mice, vaccination with radiation-attenuated (RA) cercariae induces significant levels of resistance to S. japonicum challenge in rats, rabbits, sheep, and bovines (reviewed in Ref. 46). Studies using B cell–deficient and cytokine-deficient mice demonstrated that successful antischistosome vaccination required induction of strong Th1 and Th2 responses. Following infection by normal or RA cercariae, the predominant early immune response was Th1-mediated and aimed at the adult worm. Following egg deposition in tissues (6-week post infection for S. mansoni or 4–5 weeks for S. japonicum), the Th1 response is diminished, being replaced by a prominent Th2mediated phase. Indeed, it appears that egg antigens are able to directly suppress the Th1 response (28,47), a phenomenon that may also occur in humans. The Th2 response results in an increase in serum IL-5, eosinophilia and a granulomatous response aimed at the egg, resulting in collagen deposition, tissue fibrosis, and the disease manifestations of schistosomiasis. The precise role of eosinophils in the disease process in the mouse model of infection remains undetermined (48). As referred to earlier, in the case of S. japonicum, zoonotic transmission adds to the complexity of control programs, but provides a unique opportunity to develop a transmission blocking veterinary vaccine to help prevent human infection and disease. However, studies of protective immunity in bovine schistosome infections are few (24), and consequently, our knowledge of the immunology of schistosome infections in buffaloes and cattle is extremely limited. This is particularly the case for water buffaloes where immunological reagents for studying immune responses are scarce. Recent PZQ treatment and reinfection studies of bovines infected with S. japonicum in China have indicated that age-related resistance occurs in buffaloes but not cattle (49). Whether this self-cure phenomenon has an immunological basis has yet to be determined. Additional studies on the immunology of buffaloes and cattle represent an important area for future research and will be essential in selecting S. japonicum vaccine antigens and defining the optimum route of immunization.

HUMANS AND RESISTANCE TO SCHISTOSOMIASIS Numerous longitudinal cohort studies of reinfection rates following curative drug treatment have shown that people living in schistosome-endemic areas acquire some form of protective immunity after years of exposure to S. mansoni, S. haematobium, or S. japonicum (1,8,10,13,50). However, age-related innate resistance mechanisms may also play an important part in the epidemiology of schistosomiasis (1,51). Immune correlative

studies in various parts of the world suggest that acquired antischistosome protective immunity after curative drug therapy is mediated (although not exclusively) by a Th2 response, orchestrated by IgE against adult and larval antigens, which stimulate eosinophils to release cytotoxins targeting schistosomula (1,12,13). Despite the protective role of IgE, high levels of IgG4 are also produced during infection, potentially blocking the protective effects of other immunoglobulins, suggesting that immunity to reinfection might be more closely related to the IgE/IgG4 balance than to the absolute level of each isotype (52). The development of a vaccine for schistosomiasis that is dependent on IgE would be potentially problematic, and would certainly be impeded by regulatory and safety issues. Therefore, looking to the immune responses of chronically infected individuals, and even those who become refractory by producing IgE after drug treatment, should be approached with caution. Perhaps the most important findings yet toward understanding protective immunity to schistosomiasis come from studies on individuals (particularly those from Brazil) who display a naturally acquired immunity in the absence of prior drug treatment (53–55). These small (less than 1% of exposed individuals) but well-defined cohorts are referred to as endemic normals (53), or more recently, putative resistants (PRs) (56). These individuals are resistant to infection despite years of exposure to S. mansoni, and are defined as (i) negative over five years for S. mansoni infection based on fecal egg counts, (ii) never treated with anthelmintic drugs, (iii) continually exposed to infection, and (iv) maintain a vigorous cellular and humoral immune response to crude schistosome antigen preparations (53–55,57). PR individuals mount vigorous but very different (to chronically infected patients) immune responses to crude S. mansoni extracts from schistosomula (using detergent to solubilize the tegument) and adult worms (54,55,58). In response to stimulation with these antigens, peripheral blood mononuclear cells from PR individuals secrete both Th1- and Th2-type cytokine responses (58,59), while chronically infected individuals make a Th2-type response (60). It is the Th1 response (particularly IFN-g) to schistosomula antigens that is thought to be the key to resistance to schistosomiasis in these subjects (53). Indeed, recent studies described the use of PRs to select two new vaccine antigens that are expressed in the tegument membrane of S. mansoni, Sm-TSP-2 (56), and Sm29 (61). Both proteins were preferentially recognized by sera from PR individuals as opposed to sera from chronically infected patients, supporting the use of sera (and cells) from PR individuals to guide discovery of new vaccine antigens, particularly the apical membrane proteins of the tegument. Human immunity to S. japonicum has predominantly been assessed by reinfection and immune-correlative studies. As in S. mansoni and S. haematobium, acquired immunity to S. japonicum develops with age, but there is also evidence that host factors, including pubertal development, mediate, in part, resistance to infection and reinfection with S. japonicum (62,63). Like the African schistosomes, a high IgG4/IgE ratio to adult worm antigen (AWA) and soluble egg antigen (SEA) correlates with susceptibility to reinfection, whereas IgE excess correlates with resistance to reinfection (64). Further, peripheral blood mononuclear cells taken from resistant individuals in China produced significantly greater amounts of IL-10 in response to parasite extracts and recombinant antigens in vitro (65). Similar field-based studies in the Philippines suggested that Th1-type

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responses, typified by IFN-g responses to AWA and recombinant paramyosin appeared to be important in predicting resistance. Moreover, an IgE response to AWA among females less than 20 years (5 to 19 years) and IgA responses to SWAP in younger (5 to 19 years) age groups were associated with lower worm burdens. Compatible with other studies on human schistosomes, IgM reactivities to AWA and SEA in the 5- to 19-year-old age group predicted susceptibility to infection as did IgG4 responses to recombinant paramyosin (66,67).

DEVELOPMENT OF RECOMBINANT VACCINES Schistosomes do not replicate within their mammalian hosts. Consequently, a non-sterilizing, naturally or vaccine-acquired immunity could significantly decrease human pathology and disease transmission. Vaccination against schistosomes can be either targeted toward the prevention of infection and/or to the reduction of parasite fecundity. A reduction in worm numbers is the gold standard for antischistosome vaccine development with the migrating schistosomulum stage likely to be the major vaccine target of protective immune responses. However, as schistosome eggs are responsible for both pathology and transmission, a vaccine targeting parasite fecundity and egg viability is appropriate, although the generation of a vaccine-induced immune response against the egg stage needs to be carefully considered from a pathogenesis perspective. While regularly inducing 50% to 70% (over 90% in some cases) protection in experimental animals and additional immunizations boost this level further, it may be premature to pursue RA schistosome vaccines for human use, but their development for veterinary application is feasible. The concept is proven and many of the requisite techniques, although requiring refining and upscaling, are published. Although technically challenging, there is a case for promoting the development of a live, attenuated and cryo-preserved schistosomulum vaccine for use against S. japonicum in buffaloes to reduce zoonotic transmission to humans in China (21). If successful, the veterinary vaccine could provide a paradigm for the development of antischistosome vaccines for human use. While the efficacy of the S. mansoni RA vaccine model raised hopes for the development of molecular vaccines, this has not equated to advances in the development of recombinant vaccines. Independent testing of six candidate S. mansoni antigens [glutathione-S-transferase (GST)-28; paramyosin; Ir-V5; triose-phosphate isomerase; Sm23, Sm14] in the mid1990s orchestrated by a TDR/WHO committee resulted in protective responses being recorded, but the stated goal of consistent induction of 40% protection or better was not reached with any of the antigens tested (68). Nevertheless, convincing arguments still support the likelihood that effective vaccines against the various schistosome species can be developed (18). First, as discussed above, irradiated cercariae regularly induce high levels of protection in experimental animals and additional immunizations boost this level further; second, as we have emphasized, endemic human populations develop varying degrees of resistance, both naturally and drug-induced; and, third, veterinary anti-helminth recombinant vaccines against cestode platyhelminths have been successfully developed and applied in practice (69). The optimism sparked by these arguments has resulted in the discovery of a large number of schistosome antigens (utilizing the almost complete genome sequence), and additional candidates are now being found through proteomic approaches (70,71); these two dynamic

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areas of schistosome molecular biology will be further explored below. However, antigen identification and successful protective results are of little value if recombinant proteins cannot be easily (and cheaply) produced under Good Manufacturing Practice (GMP). Even the best protective results are no guarantee for ultimate success, and the scaling-up of antigen production can be every bit as challenging as any immunological investigation. This was underscored when several of the frontline candidates chosen by the TDR/WHO committee discussed above had to be abandoned because, as well as the low efficacy recorded, hurdles in consistent protein production could not be overcome. A number of recent studies, particularly on S. japonicum (reviewed in Ref. 27), have utilized plasmid DNA vaccines to deliver protective antigens. DNA vaccines generate both T cell and B cell (or antibody mediated) immune responses, and are thus particularly appealing for schistosome vaccine development. The preparation and production of DNA vaccines is convenient and cost effective, and for use in the field, can even be used without a cold chain. Another advantage of applying DNA vaccines when compared to other approaches is the possibility of targeting the in vivo expressed recombinant antigen to different cell compartments. Furthermore, methods such as prime-boost regimens and the use of adjuvants (such as IL-12) in combination with a DNA vaccine can enhance its protective effectiveness. The advantages and disadvantages of plasmid DNA vaccination, the strategies employed for DNA vaccine delivery, and technological and clinical advances in the area have been recently reviewed (72,73).

VACCINE CANDIDATES Tables 1 and 2 summarize the data for some of the most promising S. mansoni and S. japonicum vaccine antigens discovered in the past 10 years, rather than focusing on all those (S. mansoni) that were independently tested in the mid-1990s (68), and have been extensively reviewed elsewhere (12,14,16).

Glutathione-S-Transferase Despite the discovery and publication of numerous potentially promising vaccine antigens from schistosomes, only one vaccine has entered clinical trials 28-kDa GST from S. haematobium, or BILHVAX as it is also known (12). S. mansoni GST did not obtain greater than 40% protection in the TDR/WHO independent trials (68) but numerous studies supported the developm e n t a n d e a r l y - s t a g e c li n i c al te s t i n g o f G S T as a schistosomiasis vaccine. Vaccination experiments performed with the recombinant protein in various experimental models (rodents, primates, and cattle) demonstrated a significant protective effect against schistosome infection (reduction of 40–60% of the worm burden), as well as a significant inhibitory effect on female worm fecundity and egg viability (reviewed in Refs. 11,12). The inhibition of fecundity and egg viability has been linked to inhibition of the enzymatic activity of GST (91). Phase I and II clinical trials with BILHVAX showed that the vaccine is safe and generates IgA antibodies that neutralize the enzymatic activity of GST in vitro (12). Encouraging results have been obtained with recombinant 26-kDa GST of S. japonicum, which induces a pronounced antifecundity effect, as well as a low but significant level of protection in terms of reduced worm burden. The molecule is capable of stimulating antifecundity immunity in mice (92) and pigs (53.5% decrease in liver eggs) (92) following challenge infection with S. japonicum. Similar vaccination experiments have been carried out on

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Table 1 S. mansoni Recombinant Proteins That Have Shown Vaccine Efficacy in Animal Models or Human Studies Location in adult worm

Protective role in humansb

Identity

Protective vaccine in micea

Sm-TSP-2c Tegument apical (tetraspanin D) membranec

Tetraspanin integral membrane protein

þþ worms (recombinant protein) þþ eggs (recombinant protein)

Yes—PR IgG1/G3

74 56

Sm-TSP-1

Tegument apical membrane

Tetraspanin integral membrane protein

þ worms (recombinant protein) þþ eggs (recombinant protein)

No

56

Sm29c

Tegument apical membrane

þþ worms (recombinant Unknown but has Cprotein)d terminal transmembrane domain

Yes—PR IgG1/G3

61

Sm23e

Tegument apical membrane

Tetraspanin integral membrane protein

þ worms (multiantigenic peptide – [MAP]) þ worms (plasmid DNA)

Yes—DIR IgG3 to MAP3

Sm-p80

Associated with tegument inner membrane

Calpain—neutral cysteine protease

þ worms (plasmid DNA) þþ worms (plasmid DNA including cytokines)

ND

Sm14e

Whole body, cytosolic

Fatty acid binding protein

þþ (recombinant protein)

Yes—DIR

79 80, 81, 82 77

Sm28-GSTe

Whole body

Glutathione-S-transferase

þ worms (recombinant protein) eggs

Yes—DIR

83 77

Sm28-TPIe

Unknown in adult but tegument of newly transformed somula

Triose phosphate isomerase

þ worms (transfer of anti-TPI mAb)

Yes—DIR IL-5 to MAP-4 IgG2 to MAP-4

84 76 77

Sm97 paramyosine

Tegument of Paramyosin schistosomula and musculature of adults

þ worms (recombinant and native proteins)

Yes—PR IgG DIR IgE

85 86 57 77

CT-SOD

Tegument and gut epithelia

þþ worms (plasmid DNA)

ND

87 88

Protein or cDNA

Cytosolic Cu–Zn superoxide dismutase

Reference

75, 76 77 78 71

a Data as reported in initial publications from inventor’s laboratories; þ = 30% to 50% reduction in worm/liver egg burdens; þþ ¼ 450% reduction in worm/liver egg burdens. b PR—protective role from studies with Putatively Resistant subjects; DIR—protective role from studies with Drug Induced Resistant subjects. c Identified in tegument outer membrane from biotinylated worms using proteomics (71). d Sergio Costa Oliveira, personal communication. e Vaccine efficacy tested independently (68) and adult worm reductions did not exceed 40%.

water buffaloes, the major reservoir for transmission of S. japonica in China, and only a small (but significant) reduction in worm numbers was evident in vaccinated animals (93). The clinical efficacy of BILHVAX has not yet been determined, so there is a desperate need to bring new antigens forward to clinical trials, establishing a pipeline for production and clinical assessment. Below, we highlight the most recent and pertinent data on the major vaccine antigens for schistosomiasis—some have been the focus of attention for many years, while others are newly described but show particular promise.

Tetraspanins Tetraspanins are four transmembrane domain proteins containing two extracellular loops—a short loop 1 (EC-1) with little tertiary structure and a larger 70– to 90–amino acid loop 2 (EC-2), which has four or six cysteines that form disulfide bonds. In general, the extracellular loops mediate specific protein-protein interactions with laterally associated proteins, or in some cases, known ligands (reviewed in Ref. 94). The four transmembrane domains provide stability during biosynthesis, and are crucial for assembly and maintenance of the tetraspanin web, a scaffold by which many membrane proteins are laterally organized (95). Although their functions are unknown, it is now apparent

from proteomic studies that a family of tetraspanins is expressed in the schistosome tegument (70,71,96), and at least three of these show promise as vaccines (Table 1). Sm23 was the first schistosome tetraspanin identified (97). Sm23 is expressed in the tegument of S. mansoni, and is one of the independently tested WHO/TDR vaccine candidates (68). Sm23 is most efficacious when delivered as a DNA vaccine (98), and does not confer protection as a recombinant protein when formulated with alum. S. japonicum Chinese strain SjC23 administered as a DNA vaccine in mice has provided modest reductions in worm burdens and liver eggs in some studies (99) but no protection in other labs (100). The protective effect of the SjC23 plasmid DNA vaccine was enhanced with IL-12 in pigs (101) and mice (90,99), and by CpG immunostimulatory sequence in mice (102). By combining Sj23 and Sj14 (see below section on Sm14/Sj14) as fusions or coadministered DNA vaccines, significant reductions in adult worms and reductions in granuloma sizes were achieved (103). As with the other S. japonicum candidate vaccines, extensive large animal field trials are now required to determine the precise protective potency of SjC23 with or without immunostimulatory cytokines and adjuvants. A reporter-based signal sequence capture technique was used to identify two new S. mansoni tetraspanins (Sm-TSP-1 and

Chapter 73: Vaccines Against Schistosomiasis

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Table 2 S. japonicum Protein Vaccines that have Shown Efficacy in the Mouse Model and in Reservoir Hosts of Schistosomiasis Japonica a

Antigen (native or recombinant protein)

Abbreviation

Size (kDa) Stage expressed

Biological function

Paramyosin (native)

Sj97

97

Sj97

97

Paramyosin (recombinant fragments) Triose phosphate isomerase (native) Integral membrane protein (recombinant) Aspartic protease (recombinant)

Sj97

97

SjTPI

28

Contractile protein þ others Contractile protein þ others Contractile protein þ others Enzyme

27–86 31–48 (sheep/cattle)

Paramyosin (recombinant)

Schistosomula, Adults Schistosomula, Adults Schistosomula, Adults All stages

Sj23

23

Adults

Membrane protein

SjASP

46

All stages

Calpain large subunit (recombinant) 28-kDa glutathione-Stransferase (recombinant) 26-kDa glutathione-Stransferase (recombinant) Signaling protein 14-3-3 (recombinant) Fatty acid binding protein (FABP) (recombinant) Serpin (recombinant)

Sjcalpain

80

All stages

Digestion of hemoglobin Protease

27–35 32–59 (water buffaloes/ cattle/sheep) 21–40

Sj28GST

28

All stages

Enzyme

Sj26GST

26

All stages

Enzyme

Sj14-3-3

30

All stages

Molecular chaperone

0–35 33–69 (waterbuffaloes/ sheep) 24–30 25–62 (water buffaloes/ cattle/pigs/sheep) 26–32

Sj14

14

All stage

Binds fatty acids

34–49 32–59 (rats/sheep)

Sjserpin

45

Adults

36

SjSVLBP

20

Adult males

Serine proteinase inhibitor Binds lipoproteins

SjFer

450

All stages

Iron storage

b

Very low density lipoprotein binding protein (recombinant) Ferritin (recombinant)

Worm burden reduction (%) mouse (other hosts)

20–60 17–60 (water buffaloes/ pigs/sheep) 33–77 21–24

40–41

34 35

Egg reduction (in faeces and/or liver) was also recorded with many of the candidates. When evaluated, reduced egg-hatching capacity of S. japonicum eggs into viable miracidia occurred with some vaccines. b Mucosal immunization. Source: Modified from Ref. 21, with additional data from Refs. 89 and 90. a

TSP-2) (74). Both proteins are expressed in the tegument membrane of S. mansoni (56), and TSP-2 was identified as one of only a small number of proteins that were biotinylated on the surface of live worms and subsequently identified using tandem mass spectrometry (71). TSP-2 in particular provided high levels of protection as a recombinant vaccine in the mouse model of schistosomiasis, and both proteins were strongly recognized by IgG1 and IgG3 from PR individuals but not chronically infected people (56). In addition to TSP-2, two more tetraspanins were identified from the outer tegument of biotinylated S. mansoni adults (71), and both are clearly now vaccine targets that we are exploring in our laboratories (26). The extracellular loops of TSP-2 can be expressed at very high levels in soluble and stable form in both yeast and bacterial cells (M. Tran, M. Pearson, A. Loukas, unpublished data), overcoming a major (and costly) impediment to the development of many vaccine antigens.

Sm29 and Other ‘‘New’’ Membrane Proteins Other than Sm-TSP-2, only one of the biotinylated apical membrane proteins identified by Braschi and Wilson (71) has been assessed as a vaccine—Sm29. Like TSP-2 (56), Sm29 is preferentially recognized by antibodies from PR compared with CI individuals (61). Moreover, preliminary trials in mice suggest that this protein is an efficacious recombinant vaccine [S. Costa Oliveira, personal communication; (26)], lending further support to its development as a recombinant vaccine.

Other apical membrane proteins from the tegument (71) that warrant attention as vaccines include the structural membrane proteins with large extracellular regions such as annexin and dysferlin, and other accessible (to antibodies) proteins with no homologues of known function, such as Sm200. S. japonicum expressed sequence tags (ESTs) that share greater than 50% identity to Sm29 at the amino acid level have been identified (A. Loukas, unpublished data) but have yet to be produced in recombinant form and tested as vaccines.

Calpain Calpain is a calcium-activated neutral cysteine protease. The calpain large subunit was first discovered from S. mansoni by immunoscreening of a lambda phage cDNA library with sera from infected human subjects (104). Calpain was immunolocalized to the tegument and underlying musculature of adult S. mansoni and is thought to be involved in surface membrane turnover (105). It is, however, associated with the inner tegument membrane and is not accessible to labeling with longform biotin (71). It is also present in the penetration glands and the secretions of S. japonicum cercariae (106). The large subunit of calpain, called Sm-p80, was expressed in baculovirus and the semi-purified protein induced 29% to 39% reduction in worm burdens (107). Subsequent efforts to improve the efficacy have focused on DNA vaccine constructs with and without Th1-type cytokine cDNAs (108). Calpain is also recognized as an encouraging vaccine candidate against S. japonica (109).

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Superoxide Dismutase Granulocytes release oxygen radicals that are toxic for S. mansoni, and exogenous superoxide dismutase (SOD) inhibited granulocyte toxicity for egg metabolic activity and hatching (110). A cDNA encoding an SOD with a signal peptide was cloned from S. mansoni, and its protein product was recognized by sera from infected human subjects (111). A cDNA encoding a cytosolic SOD (CT-SOD) was then identified (112) and both SODs were immunolocalized to the tegument and sub-tegumental tissues (78,88). Proteomic studies have since shown that SOD is localized below the tegument plasma membrane (70,96). Vaccination experiments using the recombinant SOD proteins has not been reported, but CT-SOD shows promise as a DNA vaccine, resulting in significant reductions in adult S. mansoni using the murine challenge model (87). While SOD homologues exist in the S. japonicum genome, they have yet to be assessed as vaccines.

Paramyosin Paramyosin is a 97-kDa myofibrillar protein with a coiled-coil structure, and is found exclusively in invertebrates. It is expressed on the surface tegument of lung-stage schistosomula in the penetration glands of cercariae (reviewed in Ref. 113) and may function as a receptor for Fc (114). The vaccine efficacy of paramyosin against S. mansoni was first described in the 1980s; mice immunized intradermally with S. mansoni extracts adjuvanted with BCG were significantly protected against subsequent infection and antibodies predominantly recognized paramyosin (115). Vaccination of mice with native and recombinant paramyosin was then shown to provide modest (26– 33%) but significant protection against challenge infection with S. mansoni (85). Native and recombinant paramyosin confer significant protection (approximately 35% decreased worm burden and 45% decreased liver egg burden) against S. japonicum in mice and buffaloes (116). There is greater than 95% homology between the paramyosin genes of S. japonicum (Chinese and Philippine strains), S. haematobium, and S. mansoni (116), and this may facilitate development of a ‘‘consensus’’ molecule as a vaccine against all three human pathogens should efficacy be improved. Currently, mathematical modeling of the likely benefits of rec-Sj-97 at its current level of efficacy as an antifecundity vaccine suggests it would prove a useful adjunct to existing control programs (8,117).

Fatty Acid Binding Proteins The S. mansoni fatty acid binding protein, Sm14, is a CT protein expressed in the basal lamella of the tegument and the gut epithelium (118). Sm14 has been thoroughly assessed as a recombinant protein vaccine and, to a lesser extent, as a DNA vaccine. Despite obtaining high efficacy with Sm14 recombinant protein in mouse vaccine trials (79), Sm14 failed to induce protection levels greater than 40% when tested in other laboratories (119) and as part of the WHO/TDR-sponsored trials (68). Coadministration of recombinant Sm14 protein with either IL-12 (119) or tetanus toxin fragment C (120) boosted protection. Immunization of mice with recombinant Sm14 expressed in Mycobacterium bovis BCG showed no induction of specific antibodies to Sm14, however, splenocytes from vaccinated mice produced IFN-g upon stimulation with recombinant Sm14. Moreover, mice that were vaccinated once with Sm14-BCG then challenged with S. mansoni cercariae showed a 48%

reduction in worm burdens, which was comparable to that obtained by immunization with three doses of recombinant Sm14 protein (80). The S. japonicum orthologue of Sm14, recombinant Sj14, also confers 34% to 49% protection in different strains of mice (121), as does an Sj14 DNA construct (103).

ANTIGEN DISCOVERY The current Schistosoma vaccine candidates may prove not to be the most effective. It is important to identify new target antigens and to explore alternative vaccination strategies to improve vaccine efficacy. The available schistosome antigens and prototype vaccine formulations induce, at best, 40% to 50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. This apparent efficacy ceiling (that also applies to antigen combinations as well) has proved a significant roadblock to success. Accordingly, the current model vaccines may not be sufficiently protective or characterized by reproducible efficacy. Difficulties in obtaining good expression levels and scaling up production according to Good Laboratory Practice (GLP)/GMP standards of the limited number of antigens selected have turned out to be another major obstacle. Some frontline candidates have suffered from difficulties in scale up production according to GLP/GMP standards and have been dropped. Feasibility of large-scale production should be a prime selection criterion in assessing the vaccine candidacy of schistosome antigens (18). Mining and functional annotation of the greatly expanded S. mansoni (122) and S. japonicum (123) transcriptomes, in combination with post-genomics technologies including DNA microarray profiling, proteomics, glycomics, and immunomics, has the potential to identify a new generation of potential vaccine target molecules that may induce greater potency than the current candidate schistosome antigens. Molecules containing signal peptides and signal anchors as predictors of excretory-secretory products, including enzymes, and components exposed on the schistosome epithelial surfaces (including receptors) that interact directly with the host immune system, are highly relevant targets for study (26,56,61,74,124). The burgeoning area of schistosome genomics and post-genomics research has been reviewed extensively (125–131). One important point that needs to be made is that the majority of studies have been undertaken on S. mansoni and S. japonicum; there is almost a complete absence of transcriptome/genome information for S. haematobium and this is clearly an important area for future study. Researchers are now armed with an almost complete genome and a well-characterized tegument proteome. It is the tegument on which many researchers have focused their efforts, but it is those few tegument proteins that are truly exposed to the host immune system in a live worm—the apical membrane proteins—which in our opinion should be a major focus for future vaccinology efforts (26). Where investigated, apical membrane proteins of the tegument have shown great promise, for example, the tetraspanins and Sm29 (Table 1). This subset of exposed proteins (70,71), which present extracellular regions of various sizes outside the cell, should attract much more attention in the future, and we advocate that efforts of schistosomiasis vaccine laboratories would be better invested in developing methods to produce and deliver schistosome surface antigens (see below) or secreted molecules, rather than continuing to identify new non-membrane spanning antigens that show modest protection at best.

Chapter 73: Vaccines Against Schistosomiasis

ANTIGEN FORMULATION AND DELIVERY Extracellular vaccine candidates need to be expressed in bacteria or eukaryotic expression systems. Many of the selected targets are likely to require processing through the endoplasmic reticulum by virtue of their expression sites in the parasite (i.e., secreted or anchored in the tegument), and this may prove challenging. An additional important consideration is that antigen identification and successful protective results are of little value if GMP cannot be applied for scaling-up of production of any vaccine candidate (18). The selection of a suitable adjuvant and delivery system to aid in the stimulation of the appropriate immune response are critical steps in the path to the development and employment of successful antischistosome vaccines, and a number of approaches have been tested with some success. Traditional approaches have seen Freund’s adjuvants used when antigens are first being assessed as vaccines in the mouse model. It must be remembered, however, that Freund’s complete adjuvant, although the mainstay of immunological adjuvants in research for decades, is not suitable for human application as it can produce a number of undesirable side effects that include the formation of local inflammatory lesions at the site of the injection that can be severe and result in chronic granulomas and abscesses. Once efficacy has been proven, other adjuvants, particularly those that are licensed (or have the potential for licensing) for human use, are used to formulate an antigen. Less conventional or less widely used approaches have been explored for adjuvanting schistosome vaccines, including live Salmonella (132), tetanus toxin (120), filamentous phage (133), recombinant Mycobacterium bovis Bacille Calmette-Gue´rin (80,134), nanoparticles (135), and various methods of mucosal delivery (136–138). Before a well-informed decision can be made on adjuvant selection, a comprehensive understanding of the desired immune response (phenotype) is necessary. This in turn implies that the immune parameters required to obtain optimal protection are known. For human schistosomiasis, this is not the case. For example, very few people develop natural resistance to the parasite in the absence of repeated anthelmintic therapy (see earlier section on PR individuals). We advocate the use of such cohorts to guide vaccine development (both antigen discovery and the phenotype of the protective response), but in reality, a schistosome vaccine will be delivered as part of an integrated control package that involves PZQ treatment before vaccination. Therefore, should we look more to the people who develop resistance to reinfection after PZQ therapy (51)? These two groups of individuals make very different immune responses to different antigens on different stages of the parasites (51,53,139). All this information is relevant, albeit complicated, when deciding how best to formulate and deliver a vaccine for human schistosomiasis. If we are to target toll-like receptors (TLRs) on antigen-presenting cells that induce a Th1 response, such as TLR-9, then adjuvants such as un-methylated CpG oligonucleotides are attractive, and although not yet widely used for schistosomiasis vaccinology, this adjuvant is showing promise for experimental vaccines against other pathogens (140). Indeed, the PR individuals identified in Brazil (53), who were utilized to identify two new tegument antigens (56,61), mounted a vigorous Th1 response to schistosomula surface antigens, making CpGs a potentially attractive adjuvant for these vaccines. CpGs are being used in conjunction with more conventional adjuvants such as alum, which induce a more Th2-like immune response. For the diphtheria-tetanuspertussis (DPT) vaccine, which is currently formulated with

815

alum, the addition of CpGs reduced the total IgE levels and increased anti-PT specific IgG2a in comparison with the ordinary DPT-alum vaccine (141). CpG-7909 has been used to improve the antibody responses generated to licensed vaccines in humans, such as the anthrax vaccine adsorbed and the Engerix-B hepatitis B vaccine (reviewed in Ref. 139). If a mixed Th1/Th2 response is optimal for a schistosomiasis vaccine, combination adjuvants such as alum-CpG seem to be a suitable way forward.

CONCLUDING COMMENTS Taking the breadth of consolidated, international efforts to generate antischistosome vaccines, there is considerable optimism that these endeavors will prove successful. In our opinion, the most recent quantum leaps forward in schistosomiasis vaccinology have been the integrated genomic and proteomic studies that have now equipped us with all the information (antigen selection at least) we need to choose the best antigens for a schistosomiasis vaccine. The recent landmark publications of the S. japonicum and S. mansoni genomes provide new avenues for antigen discovery (142,143). Again in our opinion, we emphasize that the apical membrane proteins expressed on the surface of the schistosomulum and adult worm are the logical vaccine targets on which to focus, and recent published data with some of these proteins supports this hypothesis (26,56,61). Moreover, there are mRNAs encoding novel, putatively secreted proteins without known homologues that are lodged in the tegument membrane (70,71), and these have yet to be explored. Indeed, there are very few descriptions of schistosomiasis vaccine trials with proteins that are completely unique to schistosomes and do not share sequence identity with any other proteins. When developed and employed, antischistosome vaccines will not be a panacea. They need to be regarded as one component, albeit a very important one, of integrated schistosomiasis control programs that complement existing strategies including chemotherapy and health education. Although debatable, PZQ resistance is either here or on the horizon at least, and the need for vaccines is now more pressing than ever.

ACKNOWLEDGMENTS The authors would like to acknowledge The Wellcome Trust (U.K.), The National Health and Medical Research Council of Australia, The UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and The National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S.A. for financial support of their work on schistosomiasis. They gratefully acknowledge also their group members and numerous collaborators in vaccine research.

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74 Vaccines Against Entamoeba histolytica Christopher D. Huston Department of Medicine, The University of Vermont College of Medicine, Burlington, Vermont, U.S.A.

William A. Petri, Jr. Division of Infectious Diseases and International Health, University of Virginia Health System, Charlottesville, Virginia, U.S.A.

GLOBAL IMPACT OF AMEBIASIS Amoebic colitis and liver abscess are due to infection with the enteric protozoan parasite Entamoeba histolytica. This parasite has recently been separated using modern diagnostic techniques from the nonpathogenic parasite Entamoeba dispar, which is more common and identical in appearance to E. histolytica (1). The World Health Organization (WHO) estimates that approximately 50 million people worldwide suffer from invasive amoebic infection each year, with a resultant 40 to 100 thousand deaths annually (1). Infection with E. histolytica occurs worldwide, but people living in Central and South America, Africa, and India suffer from the bulk of the morbidity and mortality (2–7). Carefully conducted serologic studies in Mexico, where amebiasis is endemic, demonstrated antibody to E. histolytica in 8.4% of the population (2). In the urban slum of Fortaleza, Brazil, 25% of the people tested carried antibody to E. histolytica; the prevalence of antiamoebic antibodies in children aged 6 to 14 years was 40% (3). An incidence of amoebic liver abscess of 21 cases per 100,000 inhabitants was observed in Hue City, Vietnam (4). A prospective study of preschool children in a slum of Dhaka, Bangladesh, demonstrated an annual incidence of E. histolytica infection in 39% of children, with 10% of the children having an E. histolytica infection associated with diarrhea and 3% with dysentery (5). The importance of amebiasis in child health may extend beyond the immediate morbidity and mortality of acute colitis or liver abscess. E. histolytica–associated diarrheal illness was negatively associated with the growth of preschool children and with their subsequent cognitive function. Children with E. histolytica–associated diarrheal illness were three times more likely to be malnourished and five times more prone to be stunted (6). Cognitive function was assessed in 191 Bangladeshi children of six to nine years using verbal and nonverbal tests. These scores were added to a health surveillance database that was compiled over the four previous years that included the incidence of diarrhea and E. histolytica infection and nutritional status. Cognitive scores were negatively associated with stunting during school age, as well as with prior E. histolytica– associated dysentery (7). Recent advances include not only the demonstration that human immunity to E. histolytica is associated with anti–galactose and N-acetyl-D-galactosamine (Gal/GalNac) lectin muco-

sal immunoglobulin A (IgA) and systemic interferon (IFN)-g, but also the identification of amoebic proteins associated with virulence, which are potential vaccine candidates (8–10). This chapter summarizes the current knowledge of how E. histolytica causes invasive disease, the role of the host innate and acquired immune responses to limiting amoebic infection, and recent progress in development of a vaccine for amebiasis.

PATHOGENESIS It is generally accepted that E. histolytica invades tissue and causes clinical disease through a well-defined sequence of events that starts with the ingestion of the infectious cyst form of the parasite from fecally contaminated food or water (11–14). Excystation of the amoebic trophozoites occurs in the intestinal lumen. The trophozoites adhere to the colonic mucus and epithelial cells through interaction of a Gal/GalNac inhibitable lectin with host Gal/GalNac-containing glycoconjugates (12,14,15). Secretion of proteolytic enzymes by the parasite may aid disruption of the intestinal mucus and epithelial barrier and facilitate tissue penetration (10). The trophozoite kills host epithelial and immune cells at points of invasion, causing the characteristic flask-shaped colonic ulcers for which it is known. Ingestion of the apoptotic corpse by the amoeba rapidly follows. Finally, E. histolytica resists the host’s immune response and survives to cause prolonged extraintestinal infection such as amoebic liver abscesses.

Adherence Several investigators have observed in vivo adherence of E. histolytica trophozoites to the colonic mucosal surface prior to tissue penetration or cytotoxicity (12–14). Adherence of the parasite to the host via a parasite lectin that binds to N-acetylD-galactosamine and D-galactose is a prerequisite for parasite cytotoxicity (12). Blockade of lectin activity with millimolar concentrations of Gal or GalNAc prevents the contact-dependent cytotoxicity for which the organism is named (15). Apoptotic death of the host cell is followed by its rapid ingestion (16). Chinese hamster ovary (CHO) cell glycosylation–deficient mutants lacking terminal Gal/GalNAc residues on N- and O-linked sugars are nearly totally resistant to amoebic adherence and cytolytic activity (17,18). Importantly, the Gal/GalNac lectin

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reduction in heterodimeric lectin and a decrease in cytotoxicity (28). The intermediate subunit is encoded by two genes, which share 81% amino acid sequence identity and are in turn members of a much larger family of amoebic genes that are transmembrane receptor kinases (22).

Invasion

Figure 1 Gal/GalNAc adherence lectin of Entamoeba histolytica. The Gal/GalNAc lectin mediates parasite adherence to, and killing of, host cells. It is present on the plasma membrane of the amoeba and is composed of three subunits. The integral membrane heavy subunit (hgl) has a short cytoplasmic tail implicated in intracellular signaling. The CRD is located within hgl. hgl is disulfide bonded to a lipid-anchored light subunit (lgl). Finally, the lipid-anchored intermediate subunit (igl) is noncovalently associated with the hgl-lgl heterodimer. The functions of lgl and igl in adherence and killing are unknown. Abbreviations: Gal/GalNAc, galactose and N-acetyl-Dgalactosamine; CRD, carbohydrate recognition domain.

also mediates adherence to human neutrophils, colonic mucins, and epithelial cells, the in vivo targets of E. histolytica (7,19). Disruption of the lectin (via inducible expression in the parasite of a dominant-negative mutant of the lectin) inhibits amoebic abscess formation in an animal model (20). The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35/31 kDa) subunits, which is noncovalently associated with an intermediate subunit of 150 kDa (Fig. 1) (12,15,20–29). The 170-kDa subunit contains a carboxyl-terminal cytoplasmic and transmembrane domain adjacent to a cysteine-rich extracellular domain (23,24). Five distinct genes (termed hgl1 to hgl5) encoding the lectin’s heavy subunit have been identified and at least partially sequenced. Ramakrishnan et al. demonstrated simultaneous expression of three different heavy subunit genes (25). At least 89% sequence homology exists within this gene family and the number and location of every cysteine residue is conserved within the regions sequenced to date (22,23,25). The carbohydrate recognition domain (CRD) is located within the cysteine-rich domain of the heavy subunit (26,27). A peptide encompassing cysteine-rich region amino acids 895 to 998 expressed in Escherichia coli has been shown to bind to Gal/ GalNAc in vitro (26). The function of the light and intermediate subunits of the Gal/GalNAc lectin remain unclear. Six to seven gene loci and at least three unique genes coding for the light subunit exist and are simultaneously expressed (25). Interference with the overall production of light subunit via antisense production results in a

After amoebic trophozoites successfully adhere to the colonic mucosa, the host may remain asymptomatic (1,30,31); alternatively, the trophozoite may spontaneously penetrate the colonic mucus and epithelial barrier in a first step toward invasive disease. Penetration to the lamina propria can occur in the absence of local inflammation. A discussion of parasite mechanisms of invasion can logically be divided into amoebic proteinases and contact-dependent cytotoxicity (32). Two classes of amoebic proteinases believed to play a role in pathogenesis have been isolated: thiol (cysteine) proteinases, which are secreted as well as located on the amoebic cell surface, and a surface-bound metallocollagenase (8). Several investigators have independently purified amoebic cysteine proteinases and confirmed their ability to degrade relevant tissue proteins in vitro, including type I collagen and the anchoring proteins fibronectin and laminin (33–35). The cysteine proteinases also degrade IgA, IgG, and the complement anaphylatoxins C3a and C5a (36–39). Cysteine proteinase activity in lysates from various E. histolytica isolates can be almost completely attributed to expression of three of these genes: EhCP1, EhCP2, and EhCP5. E. dispar carries genes homologous to four of the six genes carried by E. histolytica (termed EdCP2, EdCP3, EdCP4, and EdCP6), but appears to lack genes similar to EhCP1 and EhCP5 (40). In E. dispar, the most abundant RNA encoding a cysteine proteinase corresponds to the EdCP3 gene, which contributes very little to E. histolytica’s proteinase activity (41). Differences in the genes encoding cysteine proteinases and in their expression could partially explain the differences in pathogenicity between E. histolytica and E. dispar (41–48). Expression of the cysteine proteinases by E. histolytica is spontaneous (42), and the in vitro ability of trophozoites to degrade collagen correlates with virulence (29,39,40). Reduction in the size of amoebic liver abscesses in animal models following intrahepatic injection of virulent trophozoites confirms the role of cysteine proteinases in pathogenicity (46,47).

Cytolytic Activity The cause of the remarkable cytolytic activity for which E. histolytica is named has been the subject of intense investigation. In vitro, amoebic trophozoites incubated with human neutrophils at ratios of 1:200 remain 100% viable at three hours, while greater than 75% of the neutrophils are killed (15,48). Moments after contacting the trophozoite, target cells undergo membrane blebbing, and loss of cytoplasmic granules and membrane integrity followed within 5 to 15 minutes by cell death (15). Studies using cinemicrography show that blebbing and cell death of CHO cell monolayers occurs only upon direct contact between amoebae and their target cell (49,50). The calcium channel blockers verapamil and bepridil significantly inhibit amoebic cytotoxicity, as do the calcium chelator ethylenediaminetetraacetate (EDTA) and the putative blocker of intracellular calcium flux 8-(N,N-diethylamino)octyl-3,4,5trimethoxybenzoate (TMB-8) (49). As determined by studies utilizing the fluorescent calcium probe Fura-2 AM, the target

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cell’s intracellular calcium concentration rises irreversibly approximately 300-fold within 30 seconds of contact with a trophozoite. The addition of D -galactosamine-galactose completely blocks both this calcium flux (50) and cytolysis (15,50). Since abutment of trophozoites and target cells via centrifugation in the presence of galactose is not adequate to promote killing, the Gal/GalNac lectin appears to participate actively in the cytolytic process rather than simply bringing host cells and amoebae together (51). Upon incubation of CHO cells with purified Gal/GalNac lectin at sublethal concentrations, a reversible rise in intracellular calcium concentration of magnitude and speed comparable to that observed with whole amoebae occurs (50). Minimal chromium release from CHO cells incubated with affinity-purified lectin, however, suggests that the lectin alone is not cytotoxic; the possibility that the lectin is directly cytotoxic remains, because the purification process may alter it in ways important to cytotoxicity without altering its adherence properties (51). The ultimate cause of target cell lysis induced by E. histolytica remains unclear, and morphologic evidence suggesting both programmed cell death and/or necrosis exists (52,53). Compaction of nuclear chromatin, cytoplasmic condensation, and membrane blebbing, as well as DNA fragmentation characteristic of apoptotic cell death has been observed in a murine myeloid cell line incubated with E. histolytica. Overexpression of the Bcl-2 gene, a protein that prevents apoptosis triggered by some stimuli, did not rescue exposed cells. It is possible that E. histolytica activates a step beyond Bcl-2’s blockade of apoptosis to cause programmed cell death (52). Killing of the Jurkat human T-lymphocyte cell line occurred via apoptosis, as judged by DNA fragmentation and caspase 3 activation (54). This killing was inhibited by galactose. Classical upstream caspases seemed not to be involved, as caspase 8–deficient cells, resistant to killing by fasL, were readily killed by E. histolytica (54). Caspase 8–deficient cells treated with a caspase 9 inhibitor (Ac-LEHDfmk) (at a level sufficient to inhibit apoptosis via etoposide) were readily killed as well. In contrast, the caspase 3 inhibitor Ac-DEVD-CHO at 100 mM (sufficient to block killing via Actinomycin D) blocked E. histolytica killing, as measured both by DNA fragmentation and Cr51 release, indicating that it was necessary both for the apoptotic death phenotype and for necrosis to occur (54). Blockade of caspases has been shown to block amoebic liver abscess formation in mice (55). The mechanism by which the parasite activates caspase 3 to initiate cell death is unknown (54). Apoptotic host cells are subsequently ingested by the amoeba, an interaction that has been shown to involve exposed phosphatidylserine (PS) on the host cell surface (16,56). Several laboratories have reported the isolation of amoebic pore-forming proteins similar in function to the pore-forming proteins of the immune system (57–59). A 5-kDa polypeptide with pore-forming activity in liposomes has been described, which may be a major effector molecule mediating E. histolytica’s ability to kill endocytosed bacteria (57). The amoebapore (9) is a 77 amino acid polypeptide. Computer-aided analysis of secondary structure predicts four adjacent a-helices with tertiary structure maintained by three disulfide bonds (59). Synthetic peptides based on the amino acid sequence of the amoebapore possess cytolytic activity against bacteria and eukaryotic cells and antisense RNA inhibition of amoebapore synthesis decreased amoebic cytotoxicity (60–62).

Serum Resistance Invasion of the colon and hematogenous spread to the liver result in the continuous exposure of the extracellular trophozoite to the human complement system. The complement system is one of the first barriers to infection in nonimmune individuals; circumvention of this defense is central to the pathogenesis of amebiasis. Trophozoites activate the classical and alternative complement pathways in the absence of antiamoebic antibodies. Incubation of trophozoites in normal human sera results in depletion of human complement as measured by CH50 and C5b-9 hemolytic assays and C3 and C4 depletion (63). The amoebic 56-kDa cysteine proteinase cleaves C3 at a site one amino acid distal to that of the human C3 convertase, and may be the route by which complement is activated (63,64). Depletion of complement in hamsters by cobra venom factor-treatment increases both the frequency and severity of amoebic liver abscess, providing evidence of the protective role of the complement system in amebiasis (65). E. histolytica freshly isolated from patients with invasive amebiasis, and laboratory strains passed through animals, activate the alternative complement pathway but are resistant to C5b-9 complexes deposited on the membrane surface (62–64). On the other hand, amoebae cultured from the stool of asymptomatically infected individuals or virulent amoebae attenuated by axenic (in the absence of associated bacteria) culture activate the alternative complement pathway and are killed by C5b-9 (62–65). Killing of amoebae is mediated by the terminal complement components, and the direct lysis of sensitive but not resistant E. histolytica has been demonstrated with purified complement components C5b-9 (64–66). Resistance to terminal complement attack in E. histolytica could be due to an amoebic cell surface protein with C5b-9 inhibitory activity, or by endocytosis or shedding of the C5b-9 complex. Rapid membrane repair via shedding or endocytosis of the membrane-inserted C5b-9 complex has been postulated to confer C5b-9 resistance to several different cells, including nucleated mammalian cells and the metacyclic (infective promastigote) stage of Leishmania major. However shedding or release of C9 from the membrane does not appear to be the explanation for C5b-9 resistance in E. histolytica, as C9 binding is higher in resistant than in sensitive amoebae. Braga et al. produced monoclonal antibodies against serum-resistant amoebae and identified an antibody that increased E. histolytica lysis by human sera and by purified human complement components C5b-9. It was a surprise that the antigen recognized by the antibody was the 170-kDa lectin subunit (66). Inhibition of complement resistance by anti-170 kDa mAb was shown to be specific to mAb recognizing epitopes 6 and 7. Examination of the sequence of the 170-kDa subunit showed limited identity with CD59, a human inhibitor of C5b-9 assembly, and the purified lectin was recognized by anti-CD59 antibodies. The lectin bound to purified human C8 and C9, and blocked assembly in the amoebic membrane of the complement membrane attack complex at the steps of C8 and C9 insertion. The lectin gene family therefore appears to participate not only in adherence and host cell killing, but also in evasion of the complement system of defense via a remarkable mimicry of human CD59. Gal/GalNAc-inhibition of lectin activity had only a minor effect on C5b-9 resistance of trophozoites, suggesting that the lectin and complement regulatory domains of the lectin are distinct.

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THE INNATE AND ACQUIRED IMMUNE RESPONSE TO AMEBIASIS While the precise roles and importance of humoral and cellular responses in immunity remain to be determined, protective immunity is likely to involve elements of both. Immunization of animals with several E. histolyica antigens provides protection from an intrahepatic challenge with E. histolytica (67). These antigens include serine-rich and cysteine-rich proteins and the Gal/GalNAc adherence lectin. Antibodies appear to play a role in immunization-mediated protection, as evidenced from studies using a severe combined immunodeficient (SCID) mouse model of amoebic liver abscess. Passive transfer to SCID mice of antibodies against whole E. histolytica proteins, the serine-rich protein, or the cysteine-rich domain of the galactose lectin resulted in faster resolution of amoebic liver abscess (26,68–70). Proinflammatory cytokines also appear to be important for protective immunity. Lymphocytes from patients recovered from invasive amoebic disease proliferate in response to amoebic antigens, have amoebicidal activity, and produce interleukin-2 (IL-2) and IFN-g (71,72). Macrophages and neutrophils activated by IFN-g and tumor necrosis factor (TNF)-a are endowed with the capability of killing E. histolytica trophozoites, while in the absence of activation, these immune effector cells were killed by the amoebae (71,72). Proinflammatory cytokine production in response to E. histolytica infection may in part be an innate immune response: The purified Gal/GalNAc lectin promotes production of IL-12 and TNF-a production by macrophages (73,74). In murine macrophages TNF-g was shown to play a central role in activating macrophages for nitric oxide–dependent cytotoxicity against E. histolytica (75). Mice with targeted disruption (knockout) of either IFN-g or inducible nitric oxide synthase had more severe amebiasis, providing in vivo evidence of the importance of proinflammatory cytokines in protection (75).

ACQUIRED IMMUNITY IN HUMANS Until recently, little was known about the existence or nature of acquired immunity. The development of invasive amebiasis in some E. histolytica–colonized individuals, and documented second infections, led many to conclude that acquired immunity was nonexistent or at best incomplete (30,76–78). There was little in the way of clinical research to contradict this conclusion. Confounding the problem was the fact that most researchers failed to distinguish the invasive parasite E. histolytica from the noninvasive but identical in appearance parasite E. dispar, making their work difficult to interpret. The existence of acquired immunity to amebiasis was discovered in a prospective observational study of amebiasis in preschool children in Dhaka, Bangladesh. Immunity was linked to a mucosal antiadherence lectin IgA response. The association of mucosal anti-lectin IgA with protection was demonstrated in three ways. First, in a cross-sectional analysis, E. histolytica colonization was absent in all 64 children with stool anti-lectin IgA. Second, children with stool anti-lectin IgA acquired fewer new E. histolytica infections over a prospective period of observation [3/42 IgA (þ) vs. 47/227 IgA (); P ¼ 0.03] (Fig. 2). Finally, the appearance of a stool IgA anti-lectin response coincided with resolution of infection. Mucosal antilectin IgA is therefore an indicator of immune protection and may prove effective as a surrogate marker of vaccine efficacy (5).

Figure 2 IgA anti-CRD is associated with immunity to Entamoeba histolytica Infection. Children with fecal IgA antibodies against the Gal/GalNAc lectin CRD [IgA anti-CRD (þ); n ¼ 81] had a lower incidence of new intestinal E. histolytica infection compared with children lacking this response [IgA anti-CRD (); n ¼ 149]. The two groups were statistically significantly different (P  0.04) at every time point. The average duration of protection was 437 days (95% CI 346 to 528 days). Abbreviations: CRD, carbohydrate recognition domain; Gal/GalNac, galactose and N-acetyl-D-galactosamine; Ig, immunoglobulin. Source: From Ref. 8.

It was anticipated that IFN-g would be protective in children since it activates macrophages to kill E. histolytica and because its deficiency renders mice susceptible to amebiasis. IFN-g produced by peripheral blood mononuclear cells (PBMCs) stimulated with soluble amoebic antigen was measured in the Bangladeshi children upon entry to the study cohort. During the following three-year period of observation, 31 of the 209 enrolled children suffered from E. histolytica– associated diarrhea. Children who produced higher than the median level of IFN-g (median ¼ 580 pg/mL) had a longer survival without E. histolytica diarrhea/dysentery (logrank test P ¼ 0.03) and a reduction in the risk of E. histolytica diarrhea/dysentery by more than half (Cox proportional hazard regression ¼ 0.45; P ¼ 0.04) (Fig. 3) (79).

VACCINE CANDIDATES Although whole E. histolytica protein elicits a protective immune response indicating that an effective antiamoebic vaccine is possible, vaccines using native antigens are expensive and impractical to produce. This has driven an aggressive search for antigens that might form the basis of a cheap, recombinant vaccine. Candidate proteins (Table 1) that have been developed have mostly been cell surface or secreted. Among these are amoebic proteins implicated in pathogenesis including the Gal/GalNac lectin, the cysteine proteinases, and the amoebapore. The use of cDNA libraries has resulted in isolation of two additional candidates: the serinerich E. histolytica protein (SREHP) and the 29-kDa cysteine–rich E. histolytica antigen (79,80).

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Figure 3 High levels of IFN-g predict increased survival free of Entamoeba histolytica diarrhea. Peripheral blood mononuclear cells were stimulated with soluble amoebic extract and children grouped by IFN-g production in response to soluble amebic extract (SAE) stimulation. Children were then followed for 44 months and incidence of E. histolytica diarrhea measured. Upper line and lower line indicate children with and without IFN-g response above the median for all children (580 pg/mL), respectively. The two lines are significantly different: Logrank test P ¼ 0.03; n ¼ 92 for the low IFN-g, and n ¼ 103 for high IFN-g groups (78). Abbreviation: IFN, interferon.

PARENTERAL VACCINES The Gal/GalNac lectin plays an essential role in adherence and cytotoxicity as well as in resistance to serum complement. In addition, the lectin’s cysteine-rich extracellular domain is highly conserved (81,82). The 170-kDa heavy subunit is the predominant amoebic protein recognized by immune sera of individuals cured of invasive amebiasis from geographically diverse areas including the United States, Mexico, Africa, India, and Jordan (81,82). Greater than 90% of sera from individuals with amoebic liver abscess or asymptomatic colonization with E. histolytica contain anti-lectin antibodies (81–83). And as mentioned above, acquired immunity in humans is associated with the production of mucosal IgA against the lectin.

In one study, 100% of gerbils immunized with purified native Gal/GalNac lectin in complete Freund’s adjuvant developed high titer serum antibodies to the heavy subunit. Immune sera completely blocked amoebic adherence to CHO cells at 1/10 dilutions, and 67% of gerbils were completely protected from liver abscess following intrahepatic injection of trophozoites. Surprisingly, the remaining animals developed larger abscesses (84). Antibodies to different epitopes on the lectin’s 170-kDa heavy subunit variably enhance or inhibit amoebic adherence to CHO cells and to human colonic mucin, but no differences in the development of anti-lectin antibodies or their adherence-inhibitory properties were observed in the immunized gerbils (84). Parenteral immunization with two different recombinant peptides based on the cysteine-rich extracellular portion of the lectin’s heavy subunit has been protective in the gerbil model of amoebic liver abscess. In one study, immunization of gerbils with the recombinant LC3 region and Titermax adjuvant elicited a high titer serum IgG response capable of inhibiting amoebic adherence to CHO cells. There was a 71% reduction in the number of animals with liver abscesses following intrahepatic challenge and, in contrast to abscesses following immunization with the native lectin, abscesses in the immunized gerbils that developed them were no larger than in controls (85). Similarly, Lotter et al. immunized gerbils with several recombinant peptides based on the carboxyl-terminal portion of the lectin’s cysteine-rich extracellular domain. Immunization with a 115 amino acid peptide (termed 170CR2) completely prevented abscess development in 62.5% of animals and the remaining animals in this study developed significantly smaller abscesses than unimmunized controls. Antibody production to a 25 amino acid sequence within 170CR2 correlated strongly with development of protective immunity. Successful passive immunization of SCID mice with rabbit serum raised against the peptide reconfirmed the importance of humoral immunity in prevention of amoebic liver abscess (70). Stanley et al. identified the SREHP by screening cDNA libraries (79). This protein contains multiple tandem dodecapeptide repeats reminiscent of the repetitive circumsporozoite antigens of malarial species. Indirect immunofluorescent staining localizes the native SREHP to the cell surface and to focal areas within the cytoplasm (86). Different E. histolytica isolates have different numbers of dodecapeptide repeats encoded within their SREHP genes (87). Western blots for the presence of anti-SREHP antibodies in patients from diverse geographical regions with acute invasive amebiasis were positive in 82%. Seropositivity ranged from 65% in Durban, South Africa, to

Table 1 Known Characteristics of Current Antiamoebic Vaccine Candidates Amoebic protein

Putative function

Amoebapore Cysteine proteinase

Cytolytic activity Tissue penetration/degrades IgA, IgG, C3a, and C5a Adherence/complement resistance Possible role in adherence

Gal/GalNac lectin Serine-rich Entamoeba histolytica protein 29-kDa cysteine–rich antigen

Thiol-dependent peroxidase

Surface expression?

Conserved?

Immunogenic?

Protective in animal models?

Yes, secreted Yes, secreted

Yes Yes

Unknown Yes

Unknown Unknown

Yes Yes

Yes No

Yes Yes

Yes Yes

Controversial, probably yes

Yes

In liver abscess only

Yes

Abbreviations: Gal/GalNac, galactose and N-acetyl-D-galactosamine; Ig, immunoglobulin. Source: Adapted from Ref. 67.

Chapter 74: Vaccines Against Entamoeba histolytica

91% in Mexico City (88). Differences in the observed rates of anti-SREHP antibody production may have been due to differences in the timing of serum sampling during the course of acute illness. They also raise the possibility that local populations have differing abilities to produce anti-SREHP antibodies or that differences in the number of SREHP repeats in different E. histolytica isolates affect immunogenicity (88). Zhang et al. tested the ability of recombinant SREHP to elicit a protective immune response against amoebic liver abscess in gerbils. Gerbils were immunized either subcutaneously in a single shot or intraperitoneally in a series of three shots with a recombinant SREHP/maltose-binding protein (MBP) fusion protein combined with complete Freund’s adjuvant. Immunization completely prevented amoebic liver abscess following intrahepatic challenge in 64% of gerbils immunized intraperitoneally and 100% of gerbils immunized with a single subcutaneous shot. All of the immunized animals developed delayed-type hypersensitivity reactions (89). African green monkeys immunized with three doses of the SREHP/ MBP fusion protein developed serum antiamoebic antibodies ten days after the first booster. Unfortunately, the control monkeys in this trial did not develop liver abscesses following intrahepatic challenge, so vaccine efficacy could not be assessed (90). Screening of cDNA libraries also identified the 29-kDa cysteine–rich E. histolytica antigen, another immunogenic protein that may be suitable for inclusion in a vaccine (80). The 29-kDa antigen appears to be a thiol-dependent peroxidase, since it possesses hydrogen peroxide removing capacity in the presence of reducing agents such as thioredoxin (94,95). It may, therefore, protect E. histolytica from oxidative attack by activated neutrophils and macrophages. The location of the 29-kDa antigen within the amoeba remains controversial. Immunofluorescent staining of formalin-fixed cells with monoclonal antibodies shows the protein within both the nucleus and cytoplasm (94,95). Intraperitoneal immunization of gerbils with a recombinant fusion protein based on the 29-kDa protein and Titermax adjuvant elicited production of antigen-specific IgG and was partially protective (54% vaccine efficacy) against amoebic liver abscess following intrahepatic challenge with virulent trophozoites (96). The cysteine proteinases and the amoebapore are additional amoebic proteins associated with virulence that must be considered. Each has yet to be evaluated as a potential vaccine component. Numerous studies document the central role of amoebic cysteine proteinases in penetration of host tissues and in evasion of host defenses via degradation of IgA, IgG, C3a, and C5a. Patients with amoebic liver abscess, moreover, develop antibodies to histolysain (EhCP2), and the use of protease inhibitors in SCID mice reduces the size of liver abscesses following intrahepatic injection of trophozoites. The recombinant amoebapore’s cytotoxicity toward eukaryotic cells prohibits its use in a vaccine. Identification of antigenic regions within this peptide, however, should yield other possible vaccinogens.

ORAL VACCINES Two major oral vaccine strategies have been used: the incorporation of amoebic antigens into attenuated bacterial strains and the creation of fusion proteins composed of amoebic antigens and cholera toxin or its subunits. An effective oral vaccine against E. histolytica could have several advantages over parenteral preparations. Direct stimulation of the gut-associated

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lymphoid tissue (GALT) might stimulate production of secretory IgA more effectively than parenteral immunization, and prevent both colonization and invasive disease (97). By establishing a limited invasive infection in the host; moreover, an oral vaccine carried by an attenuated bacterial strain might provide more prolonged immunity than parenteral vaccines based on the same antigens. Combination vaccines providing protection against multiple organisms may also be possible. For example, immunization with attenuated Salmonella typhi strains engineered to express amoebic antigens might protect against both amebiasis and typhoid fever. Finally, the lower cost and ease of administering an oral vaccine would increase acceptance in developing nations. An oral attenuated vaccine for typhoid fever is currently in use in humans. Foreign antigens expressed in attenuated Salmonella species can effectively stimulate both cell-mediated immunity and production of secretory IgA (98). Oral immunization of mice and gerbils with an attenuated strain of Salmonella typhimurium that expresses the SREHP/MBP fusion protein at high levels resulted in production of secretory IgA and serum IgG. Anti-lipopolysacharide (LPS) antibodies also developed in both sham immunized and immunized animals, suggesting that the amoebic antigen did not impair the immune response to the Salmonella infection. A vaccine protective against both, therefore, might be possible. Following intrahepatic injection with amoebic trophozoites, 100% of control gerbils and only 22% of immunized gerbils developed abscesses in this study (99). In another study, oral immunization of gerbils with Salmonella dublin expressing a fragment of the Gal/ GalNac lectin resulted in significant reduction in mean abscess weight, but no significant difference in the number of animals developing abscesses. No serum antiamoebic antibody production was observed in this study, suggesting that the observed protection may have been cell mediated (98). The plasmid carrying the lectin fragment, however, was somewhat unstable in vitro; higher or more prolonged expression of the antigen may have resulted in antibody production and in greater vaccine efficacy (98). Cholera toxin has two subunits, a 28-kDa A subunit with ADP-ribosylating activity, and an 11.5-kDa B or binding subunit. The A subunit contains A1, the active toxin domain, and A2, which noncovalently links subunit A to five B subunits. A pentamer of B subunits binds the intestinal epithelium. Whole cholera toxin stimulates production of serum IgG and secretory IgA when orally administered, and also stimulates immunity to coadministered antigens (100). In humans, the B subunit retains some of whole cholera toxin’s oral adjuvant properties. Parenteral immunization of rats with native Gal/GalNac lectin in complete Freund’s adjuvant followed by intra-Peyer’s patch injection of lectin with cholera toxin’s B subunit stimulates production of anti-lectin secretory IgA (101). Oral immunization of mice with the recombinant LC3 portion of the lectin and whole cholera toxin induced production of secretory IgA capable of inhibiting adherence of amoebic trophozoites to CHO cells. Interestingly, there was a negative correlation between intestinal IgA production and serum IgA and IgG titers in this study (102). High-dose oral immunization with streptococcal antigens by other investigators has resulted similarly in a strong mucosal immune response with no systemic antibody production, while lower doses led both to mucosal and systemic antibody production. A potential limitation of strategies combining recombinant peptides with cholera toxin’s B subunit is that large

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Figure 4 Intestinal inflammation in the murine model of amoebic colitis. (A) Mucosal hyperplasia and submucosal infiltration (40, H&E). (B) Trophozoites ulcerating the epithelium and occupying the lumen (1000, H&E) (106). Abbreviation: H&E, hematoxylin and eosin.

attached molecules might prevent pentamerization of the B subunit and reduce its adjuvant properties by changing its ability to bind to intestinal epithelium. Coupling of antigens to the B subunit via A2 to create holotoxin-like molecules could potentially increase their immunogenicity as well as facilitate the use of larger recombinant antigens. A holotoxin-like molecule containing the SREHP fused to the A2 domain of cholera toxin (SREHP-H) has been created. Oral immunization of mice with SREHP-H coexpressed with the cholera toxin B subunit in E. coli resulted in production of mucosal IgA and serum IgG antiamoebic antibodies (103).

A MURINE MODEL OF INTESTINAL AMEBIASIS DEOMONSTRATES EFFICACY OF GAL/GALNAC LECTIN VACCINATION A major advance in vaccine development has been the development of a murine model of intestinal amebiasis (Fig. 4) (104). The C3H and CBA strains of mice develop a nonhealing amoebic colitis when trophozoites are injected into the cecum. We tested if vaccination with the E. histolytica Gal/GalNAc lectin could prevent cecal infection and subsequent colitis in the C3H mouse model. Vaccination prevented intestinal infection with efficacies of 84% and 100% in the two trials. Mice with detectable prechallenge fecal anti-lectin IgA responses were significantly more resistant to infection than mice without fecal anti-lectin IgA responses. These results show for the first time that immunization with the Gal/GalNAc lectin can prevent intestinal amebiasis in mice and suggest a protective role for fecal anti-lectin IgA in vivo (105).

CONCLUSIONS AND AREAS FOR FURTHER INVESTIGATION The identification of acquired immunity to amebiasis in humans lends tremendous credence to the development of a vaccine against E. histolytica. Many obstacles to the production of a vaccine have fallen by the wayside in the last decade. Effective immunity in humans is now known to be associated with a mucosal IgA response against the Gal/GalNAc lectin, one of several well-characterized virulence factors of the parasite, and an acquired systemic production of IFN-g. A mouse model of amoebic colitis has been developed and allows for the first time identification of protective immune responses at the mucosal surface where infection occurs. Well-characterized

cohorts of children with E. histolytica infection have been described. The extremely high rates of new E. histolytica infection in these children should enable the testing of vaccine efficacy using small numbers of patients. Although much progress has been made, the burden of disease due to amebiasis throughout the tropical and subtropical world makes vaccine development an urgent task.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant AI26649 to W.A.P. and AI072021 and P20 RR021905-01 to C.D.H.

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75 Hookworm Vaccines Jeffrey M. Bethony, Maria Elena Bottazzi, Ami Shah Brown, David Diemert, and Peter Hotez Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University and Sabin Vaccine Institute, Washington D.C., U.S.A.

Alex Loukas Queensland Institute of Medical Research, Queensland, Australia

INTRODUCTION In the poorest rural areas of sub-Saharan Africa, Asia, and the tropical regions of the Americas, between 576 and 740 million people live with adult hookworms in their small intestine (1,2). The most common hookworm is Necator americanus, which probably accounts for more than three-quarters of the world’s cases, followed by Ancylostoma duodenale and A. ceylanicum (3). When an individual is parasitized with 25 N. americanus adult hookworms, an estimated 1 mL of blood will be lost daily into the host gastrointestinal tract. When we consider that this amount of blood contains 0.55 mg of iron, roughly equivalent to the daily iron requirements of a child and possibly more for a pregnant woman in the tropics (4), the health consequences of chronic hookworm infection can be devastating. As host iron stores are depleted, the continued blood loss from hookworm infection can lead to both iron deficiency anemia (IDA) and protein malnutrition, which is referred to as hookworm disease. Because children and pregnant women are the most likely to have low iron reserves in developing countries, they are considered the groups at highest risk for acquiring hookworm disease. In children, hookworm disease is associated with severe deficits in development, especially physical growth retardation, reduction in intelligence, memory, and cognition (3). In pregnant women, hookworm disease results in high maternal morbidity and mortality, and subsequently, low neonatal birth weight and diminished infant survival (5,6). Through such mechanisms, hookworm infection accounts for the annual loss of approximately 22.1 million disability adjusted life years (DALYs) (7), making hookworm second only to malaria as the most important parasitic infection of humans (Table 1). In addition to its health-related effects, hookworm infection and disease produce serious socioeconomic consequences. Through its effects on child development hookworm infection causes significant reductions in educational performance and school attendance (8). Possibly, these educational effects account for the estimation that chronic hookworm infection in childhood reduces future wage earning capacity by 43% (9). In this way, hookworm, like other neglected tropical diseases, promotes poverty in the developing world (10) so that the control of hookworm infection would represent an important step toward attaining several Millennium Development Goals (MDGs) for sustainable poverty reduction by 2015 (11).

Since 2001, when the 54th World Health Assembly adopted a resolution to implement child-deworming programs in endemic countries, this has become the major approach to morbidity control for pediatric helminth infections. Deworming, which is typically carried out with a single dose of the anthelminthic drugs albendazole or mebendazole to simultaneously target hookworm and other soil-transmitted helminth infections (e.g., ascariasis and trichuriasis), has been shown to lead to improvements in child growth, development, and education, as well as improved pregnancy outcome when used during the antenatal period (5,6). However, concerns regarding the sustainability of deworming because of lower than expected efficacies of single-dose anthelminthics, especially with mebendazole, together with high rates of hookworm reinfection following treatment, and the potential for emergence of anthelminthic drug resistance have prompted the search for alternative control tools (12,13). The development of a safe and effective vaccine to prevent hookworm infection would therefore represent both an important public health breakthrough as well as a potentially effective poverty reduction measure (14). Ultimately, the successful development of a safe and effective human hookworm vaccine (‘‘the HHV’’) would represent a critical tool for achieving MDGs related to child and maternal health, poverty and hunger, and MDG 6, which is ‘‘to combat HIV/AIDS, malaria and other diseases’’ (11).

EPIDEMIOLOGY, GEOGRAPHIC DISTRIBUTION, AND DISEASE BURDEN OF HOOKWORM INFECTION Essential Elements of Hookworm Epidemiology Hookworms do not replicate within the human host, and the morbidity of hookworms as well as other helminths is related to the number of worms causing blood loss. This parameter is typically expressed as ‘‘intensity of infection’’ and is commonly measured by the number of eggs per gram (EPG) of feces. On the basis of EPGs, individuals are then classified into categories of ‘‘light,’’ ‘‘moderate,’’ and ‘‘heavy’’ infection by the World Health Organization (WHO). Heterogeneity in intensity of infection is a hallmark feature of hookworm epidemiology, so that approximately 70% of the worm burden occurs in 15% of infected individuals (15–17). Those with moderate and high

Chapter 75: Hookworm Vaccines Table 1 Ranking by DALYs of Selected Infectious and Tropical Diseases Disease condition

Disease burden (DALYs) (million)

Lower respiratory infections HIV-AIDS Diarrheal diseases Malaria Tuberculosis Hookworm infection Measles Lymphatic filariasis Schistosomiasis Leishmaniasis

91.3 84.5 62.0 46.5 34.7 22.1 21.4 5.8 4.5 2.1

Abbreviation: SALY, disability adjusted life years. Source: From Ref. 32.

intensity hookworm infections are at the greatest risk of developing hookworm anemia (3). Age is an important risk factor for heterogeneity. Hookworm infection intensity exhibits a unique age distribution among the soil-transmitted helminth infections and peaks in adulthood, unlike ascariasis and trichuriasis, which intensity peaks in childhood (18–20). Evidence also exists for household and micro-geographical clustering of hookworm-infected individuals (21), and there is evidence that hookworm intensity may carry a heritable pattern (22). Finally, an increasing number of studies have shown that hookworms commonly coinfect individuals with other helminths, especially schistosomes (23,24). Geographic Distribution The geographic distribution of hookworm infection reflects two major elements: poverty and an appropriate climate and ecology. The basis for the link between poverty and hookworm infection was reviewed recently (10), and includes associations between increased transmission and inadequate sanitation, including poor housing construction (e.g., dirt floors), as well as a lack of access to essential medicines, especially anthelminthic drugs. Among the important factors related to climate and ecology are high surface temperature (15,25), altitude, soil type, and rainfall (26,27). Today, the greatest number of hookworm cases occur in sub-Saharan Africa (198 million cases), followed by Southeast Asia and the Pacific region (149 million), India and South Asia (130 million), Latin America and the Caribbean (50 million), China (39 million), and the Middle East (10 million) (1,2). With the exception of the Middle East, some areas of China, north of the Yangtze River, northern India, and restricted geographic regions of Africa and South America where A. duodenale is found, N. americanus is the predominant hookworm in all these regions. In some areas, mixed infections occur (3). Disease Burden Studies by Stoltzfus and colleagues (28) reveal that hookworm accounts for a significant percentage of the anemia disease burden in developing countries. Among some populations of sub-Saharan Africa, hookworm infection in children was shown to account for up to 41% of the IDA and 57%of moderate to severe anemia (28); it is also an important cause of anemia in Brazilian (29) and Southeast Asian children. Brooker et al. (30) have recently completed a meta-analysis that confirms that among the estimated 44 million pregnant women infected with hookworms (31), this infection is a major cause of anemia.

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Although hookworm is not considered a significant cause of mortality in developing countries, such estimates of the contribution of hookworms to anemia translates into a significant impact on global morbidity. Current DALY estimates for hookworm vary widely, ranging from 1.8 to 22.1 million DALYs lost annually (1). The lower estimate is roughly equivalent to that of otitis media whereas the higher estimate suggests that the disease burden of hookworm infection is approximately onehalf that of malaria’s (3,32). Such variation largely reflects significant differences in assumptions regarding the contribution of hookworm to IDA and protein malnutrition in developing countries (32). However, new meta-analyses for the contribution of hookworm to both childhood and maternal anemia (30) are expected to result in a revision of the DALY estimates caused by hookworm infection.

NATURAL HISTORY AND IMMUNOLOGY OF HOOKWORM INFECTION The life cycle of N. americanus and A. duodenale have been reviewed previously (3). Briefly, humans become infected with hookworms when third-stage infective larvae (L3) penetrate through the skin and then migrate into subcutaneous venules and lymphatics before being swept via the afferent circulation and entering the pulmonary capillary bed. From there, the L3 enter the respiratory tree through the alveolae and ascend the bronchioles, bronchi, pass over the epiglottis, and enter the gastrointestinal tract. In the small intestine, the L3 molt twice to become adult male and female hookworms where they can live for five years or more. The adult worms, approximately one centimeter in length, attach to the mucosa and submucosa, lacerate capillaries and arterioles, and then feed on host blood and mucosal tissues. The hookworms mate and produce thousands of eggs that exit the body in the feces. The eggs hatch in soil with adequate moisture and high temperatures and then molt twice to the L3 stage, which seeks higher ground to come into human skin contact. A. duodenale is also orally infective. The immunology of human and animal hookworm infection is complex and has been the subject of several recent papers and reviews (33–36). Briefly, with respect to animal hookworm infections, there are three major laboratory models for studying host immune responses. Unfortunately, no single model adequately or completely reproduces the immune responses to human hookworm infection. Of all of the animal models in current use, A. caninum infections in dogs most resemble human hookworm infection, with respect to the ability of adult hookworms to live the longest and in terms of the relationships between number of hookworms and fecundity and blood loss (35). Described below is the use of attenuated and irradiated L3 (irL3), which were used to develop a canine vaccine against hookworm infection. The golden hamster (Mesocricetus auratus) has also been used for both A. ceylanicum and N. americanus infections, both of which are characterized by host blood loss, lymphoproliferation, and host anitbody production during infection (37–39). However, the model also has limitations for purposes of vaccine testing as frequently less than 10% to 20% of the L3 develop to adult hookworms in the hamster gastrointestinal tract (38). Most of the knowledge of human immunity to hookworms relies on immunoepidemiologic studies of hookworminfected humans residing in endemic areas (36). These studies are hampered by difficulties in following untreated patients over long periods of time (36). Another valuable source of

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information has been the study of the human immune response to hookworm infection following anthelminthic treatment. In addition, there are some published studies on human volunteers who were infected with controlled low-dose hookworm infections (36). Naturally infected humans mount antibody responses involving all isotypes and IgG subclasses, with IgE exhibiting the greatest increases compared with the others (1). Most infected people acquire a mixed Th1/Th2 cell response in which both types of cytokines are produced (33,36,40–42). However, there is also evidence that Th2 responses are associated with protective immunity, including an important observation made between IL-5 secretion and reduced hookworm burden (33,36). Th2 responses have also been linked to protection against animal hookworm infections (43) and antibodies to either irL3 or selected hookworm antigens for example, ASP-2 (Ancylostoma secreted protein 2) and Ac-16 (also known as SAA-2, surface associated antigen 2) have been shown to reduce larval entry through skin in vitro (44,45). Complicating this immunological picture is the observation that hookworms can live for years in the host gastrointestinal tract through the processes of immunomodulation. Several anti-inflammatory, immunosuppressive, and immunomodulatory polypeptides have now been identified and cloned from hookworms (reviewed in Refs. 15 and 46). Parasiteinduced host immunosuppression or immunomodulation may partly explain the epidemiological observation that in endemic regions, both the prevalence and intensity of hookworm infection increases with age (18). Through immunomodulation, it has also been speculated that hookworm and other helminth infections may adversely influence host immune responses to malaria and other infections (47,48). The existence of hookworm-mediated immunosuppression has practical implications for vaccine development. Because vaccineinduced immunity is potentially susceptible to immunosuppression, protective responses may not develop unless preexisting infections are removed by chemotherapy. This observation is supported by studies in hamsters infected with A. ceylanicum (49).

HUMAN HOOKWORM VACCINATION The development of a recombinant hookworm vaccine is based on the proof of principle established by the canine irradiated larval vaccines developed during the 1960s and 1970s, together with evidence that adult worms could also be targeted by immunologically interfering with parasite-specific molecules involved in blood feeding (reviewed in Ref. 50).

Targeting Larval Antigens—The Canine Irradiated Larval Vaccine Paradigm During the 1930s and 1940s, a vaccine containing live A. caninum larvae was shown to protect laboratory dogs against challenge infections (reviewed in Refs. 51 and 52). Although sterilizing immunity was not achieved, vaccinated dogs did not develop anemia despite receiving challenges of several thousand L3. A commercial vaccine consisting of irradiation-attenuated L3 (irL3) was later developed, which also resulted in significant reductions in hookworm burden after challenge infection (53,54). This vaccine was marketed for two years in the United States during the 1970s (54). Dr. Thomas Miller who led the development of a commercial canine irL3 vaccine (which was ultimately discontinued as a commercial product)

hypothesized that protection against challenge infection of vaccinated pups was attributable to reduction in worm burden from larval infectivity, reduction in blood loss because of worm burden reductions, and a sterilizing effect on female worm fecundity as seen in reduced EPGs (52). These observations, together with earlier studies showing the importance of larval antigens in mediating protective immunity, stimulated an antigen discovery program that set out to identify the key hookworm L3 secreted and surface proteins as targets for an HHV(reviewed in Refs. 50 and 55). To assess the vaccine efficacies of many hookworm recombinant antigens, which have been cloned and expressed beginning in the 1990s (15), a scoring system was developed that incorporated essential criteria for determining vaccine efficacy, including reductions in worm burden, host blood loss, EPGs, and immunoepidemiological criteria (52). On the basis of this ranking system, the larval antigen ASP-2 was selected as a lead recombinant antigen for further process development, current good manufacture practices (cGMP) manufacture and clinical testing (Table 1). ASP-2 belongs to the pathogenesis related protein (PRP) superfamily (50,55–57). Among the studies pointing to the efficacy of ASP-2 was the immunological recognition of this antigen by sera from dogs vaccinated with irL3 (45), the ability of sera from dogs vaccinated either with irL3 of A. caninum or recombinant ASP-2 to inhibit larval migration in vitro (45,57), animal protection experiments conducted in dogs and hamsters challenged with A. caninum (45) or A. ceylanicum (58,59) and N. americanus (39), respectively, and human immunoepidemiological studies (45). For similar reasons, an L3 surface protein that immunolocalizes to the parasite cuticle and hypodermis, and known as SAA-2 (surface associated antigen 2) was also selected for further process development and manufacture (44).

Targeting Adult Blood Feeding Through Specific Antigens To complement the larval antigens, a second approach to hookworm vaccination was also developed. This second path involved identifying key adult hookworm proteins involved in blood feeding at the site of parasite attachment in the gastrointestinal tract. Vaccination with an adult antigen would reduce both blood loss and EPGs. Adult hookworm blood feeding relies on ordered processes reviewed previously (52), including lysis of ingested red cells, and step-wise hemoglobin breakdown through the action of parasite-derived hemoglobinases. Vaccination of dogs with recombinant enzymes involved with blood feeding provided evidence that it is feasible to interfere with blood feeding pathways as a strategy for vaccination (52,60,61). To date, the most promising preclinical data has been obtained with the cathepsin D-like aspartic protease APR-1 and a glutathione-S-transferase known as GST-1 (Table 2). Recombinant APR-1 from both A. caninum (Ac-APR-1) and N. americanus (Na-APR-1) cleaves host hemoglobin at a critical juncture that unravels the hemoglobin tetramer, thereby rendering it susceptible to attack by other degradative enzymes (52). Vaccination of dogs with recombinant Ac-APR-1 induced immune responses that resulted in significantly reduced hookworm burdens and fecal egg counts (52,61). The vaccinated dogs were not only protected from blood loss and anemia, but in addition, it was shown that immunoglobulin from the vaccinated dogs directly inhibited enzymatic activity of the parasite (52). Another family of adult worm proteins under active development as recombinant

Chapter 75: Hookworm Vaccines Table 2 Major Hookworm Antigens Under Development by the HHVI-Sabin Candidate antigen

Molecular weight (kDa)

Life cycle stage or target

Stage of product or clinical development

Na-ASP-2 Na-APR-1 Na-GST-1

21 48 24

L3 L3 and adult Adult

Na-GST-1APR-1 Chimera

25

None

Phase 1 Preclinical cGMP manufacture Preclinical

vaccines are the GSTs. These enzymes might act at the tail end of the blood-feeding cascade, potentially neutralizing the toxic by-products of heme after its release from digested hemoglobin. Ac-GST-1, a novel GST from A. caninum, possesses a novel high-affinity binding site for hematin (62). The potential role of Ac-GST-1 in heme detoxification prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs or hamsters with Ac-GST-1 resulted in high levels of worm burden and egg count production following larval challenge (39,62). The Na-GST-1 orthologue from N. americanus has now been cloned and expressed at high yield in yeast, and also results in protection in hamsters (63). Either Na-GST-1 or NaAPR-1 will be selected for process development before entering pilot cGMP manufacture and phase 1 clinical testing. A chimeric molecule comprised of components of both antigens is also being developed.

The Bivalent Human Hookworm Vaccine Ultimately, it is envisioned that an efficacious hookworm vaccine will be bivalent and comprised of larval or adult antigens. The HHV could consist of a protein that targets both invasion and migration of the L3 stage hookworm and blood feeding by the adult stage hookworm.

FROM THE BENCH TO THE BUSH: PROCESS DEVELOPMENT AND TECHNOLOGY TRANSFER The HHV Initiative is a nonprofit product development partnership of the Sabin Vaccine Institute (HHVI-Sabin), created in collaboration with research, development, cGMP manufacturing, and clinical testing units located at George Washington University (Washington D.C., U.S.A.), Queensland Institute of Medical Research (Brisbane, Australia), London School of Hygiene and Tropical Medicine (London, U.K.), Oswaldo Cruz Foundation (FIOCRUZ, Belo Horizonte, Rio de Janeiro, Brazil), and Instituto Butantan (Sao Paulo, Brazil). Founded in 2000, the HHVI-Sabin is funded through support of the Bill and Melinda Gates Foundation as well as other donors. The current product development strategy of the HHVI-Sabin centers on the development and clinical testing of a bivalent injectable product comprised of a recombinant protein antigen from the infective larval stages of N. americanus and a recombinant protein antigen from N. americanus adult hookworms. The adjuvant for the HHV will be comprised of Alhydrogel1. Following additional clinical testing, the deoxyoligonucleotide adjuvant CPG 10103 (licensed from Coley Pharmaceuticals, Wellesley, Massachusetts, U.S.) may be added to the HHV.

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The philosophy of the HHVI-Sabin is to conduct staggered development for the two lead larval and two lead adult vaccine antigens. In the event that one fails in early product or clinical development, the HHVI-Sabin can rapidly transition to several backup antigens. Early development of the vaccine candidate antigens started with the selection of a suitable expression system. Through these efforts, hookworm antigen sequences were cloned into suitable expression vectors, screened, and tested for small-scale expression. These findings led to the next step in early development, which was to optimize expression and purification processes that were easily scalable, of high yield, and would deliver high purity and consistent product. To date and for each of the vaccine candidate antigens, the HHVI-Sabin has developed the basic processes and procedures to be used in manufacture and production of both drug substance (recombinant proteins) and drug product (vaccine). Multiple fermentation parameters have been evaluated up to the 10 L scale, with the aim of providing evidence of reproducible high-yield production. As done for fermentation, purification processes started at the small scale, and these were developed with the aim of keeping costs as low as possible. The costs of various chromatographic resins can vary substantially, and this should factor into the design of purification processes. Nonetheless, issues of price may be less important if a given column resin is required for optimal yield and purity of the target protein, such that the value added by improving yield offsets the price of expensive columns. Furthermore, both process development and downstream manufacturing rely on the development of assays for the evaluation of the product’s identity, color and appearance, purity, and antigenicity and thermostability. These assays have to be developed and qualified for their sensitivity, specificity, accuracy, and reproducibility.

Process Development Status of L3 Hookworm Antigens Of the 11 major L3 recombinant antigens discovered, cloned, expressed, and pre-clinically evaluated by HHVI-Sabin, only one these candidate antigens, Na-ASP-2, has met sufficient criteria to advance it into clinical development, pilot cGMP manufacture, and phase 1 clinical testing. The Na-ASP-2 hookworm vaccine is comprised of recombinant Na-ASP-2 protein formulated with Alhydrogel in a buffer. The purified recombinant protein has a molecular mass (confirmed by mass spectrometry) of 21.3 kDa (197 amino acids), with an N-terminal six amino acid vector tag (EAEAEF) to facilitate expression, and a single O-linked mannose (57). Evidence that the recombinant protein accurately reproduces the folding of the native Na-ASP2 includes studies showing that antibody directed against the recombinant protein recognized the native protein on western blots, and that antibody prepared against the recombinant protein inhibited larval migration in vitro (57) as well as immunprecipitation studies done during phase 1 clinical testing (64). The buffer originally used was comprised of phosphate buffered saline at physiological pH, but has since been changed to sodium acetate buffer (pH 6.0). The Na-ASP-2 hookworm vaccine has been produced as a sterile, injectable vaccine under cGMP conditions and tested with qualified analytical methods for identity, purity, and antigenicity and, in animals, for potency. A nonclinical toxicology study in rats was completed without apparent safety issues. Following submission of an IND application to the U.S. FDA in

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December 2004, a phase 1 study was successfully completed in healthy, hookworm-uninfected adults in the United States (64).

Process Development Status of Adult Hookworm Antigens Two adult antigens, Na-GST-1 and Na-APR-1, are currently in the pipeline to determine the feasibility for product development and pilot cGMP manufacture, with most HHVI efforts are currently focused on the development of the Na-GST-1. A process leading to high yields and reproducible expression of Na-GST-1 is being developed. Manufacturing and release of a cGMP lot of Na-GST-1 in yeast is currently in progress. It is expected that an IND will be submitted during 2010 to initiate a phase 1 trial in Brazil early in 2011. In addition, the expression of Na-APR-1 in both yeast and bacteria is ongoing, and plans are in place to take one of these expression systems through a program for process development, manufacture under cGMP conditions, and clinical testing (12,52).

Adjuvant Development Program The leading adjuvant technology being evaluated for the larval and adult hookworm antigens is Alhydrogel, an aluminum salt adjuvant. HHVI-Sabin is also evaluating two alternative lead adjuvant platforms including a CpG molecule and IC311, which includes a CpG molecule and an antimicrobial peptide. Other technologies that have been evaluated in animal studies include Quil A, Montanide ISA 720, Freund’s, and ISA 70.

Technology Transfer The HHVI-Sabin faces formidable manufacturing challenges for producing a product intended for the world’s poorest people. Foremost among these challenges, is hookworm’s neglected disease status, and therefore, the limited commercial market that exists for such a vaccine, making development and production by a for-profit company, such as a major vaccine manufacturer extremely unlikely. This situation has required the HHVI-Sabin to consider innovative mechanisms for largescale manufacture. In the absence of significant commercial markets, the HHVI’s strategy is to partner with vaccine manufacturers in so-called innovative developing countries (IDCs). IDCs represent middle-income countries with modest economic capacity, but with ‘‘high innovation capacity’’ (65). The HHVI has chosen Brazil as its first partner in the large-scale production of the HHV. To launch the Brazilian collaboration, a co-development agreement was signed between the HHVI and Instituto Butantan in Sao Paulo, Brazil in 2006. Instituto Butantan provides up to 80% of the vaccines and antisera that are utilized in Brazil. The HHVI-Sabin-Instituto Butantan partnership started with technology transfer for manufacture of the Na-ASP-2 hookworm vaccine. The process for manufacture of the Na-ASP-2 hookworm vaccine has been successfully transferred, and scaled-up to the 60L level at Instituto Butantan. Following this first successful technology transfer, the HHVI-Sabin is collaborating with Butantan to proceed with the scale-up production of other critical hookworm candidate antigens.

CLINICAL DEVELOPMENT OF A HUMAN HOOKWORM VACCINE The current strategy for the clinical development of candidate hookworm vaccine antigens is to first test them in uninfected, healthy adults, and then to conduct a series of age de-escalation

studies in hookworm-endemic areas, culminating in a ‘‘proof-ofprinciple’’ phase 2-study in children exposed to hookworm. Following demonstration that the vaccine has a meaningful biologic effect on hookworm infection in such a proof-of-principle study, further clinical development will be transferred to partners in IDC endemic countries with the capacity to develop and manufacture new vaccines, such as Brazil, India, or China (52,65,66). Brazil has been chosen as the first such partner, and an agreement that commits the Brazilian government to continuing the clinical development of an HHV once proof-of-principle is established in a phase 2 clinical trial has been signed, which includes the industrial-scale manufacture of vaccine and sponsorship of pivotal phase 3 efficacy studies. The first clinical trial of a hookworm vaccine candidate was a phase 1 trial evaluating the safety and immunogenicity of Na-ASP-2 in healthy adults living in the United States, who had no history of current or prior hookworm infection (64). Between 2005 and 2006, 36 adults were enrolled in this randomized, double blind, placebo-controlled study of three different concentrations of Na-ASP-2 (10, 50, and 100 mg) adsorbed to Alhydrogel. Participants received three intramuscular injections of vaccine or saline placebo on days 1, 56, and 112, and were followed until six months after the third vaccination (64). The vaccine was shown to be safe and well tolerated, with injection site reactions including mild to moderate pain, swelling, erythema, and pruritus being the most frequently observed adverse events. Additionally, vaccination induced significant anti-Na-ASP-2 IgG and cellular immune responses. Since the initial phase 1 study of Na-ASP2 adjuvanted with Alhydrogel demonstrated an acceptable safety profile and encouraging immunogenicity in an unexposed population, a second phase 1 trial of this vaccine will be initiated in healthy adult volunteers living in a hookworm-endemic area of Brazil. This first study of a hookworm vaccine in an endemic area will be a randomized, double-blind, controlled study evaluating the safety and immunogenicity of the Na-ASP-2 vaccine. The same dose concentrations and vaccination schedule will be used as in the first phase 1 trial of the vaccine. Repeating a phase 1 study in hookworm-exposed adults prior to advancing to trials in children is an important intermediate step, as neither the safety nor the immunogenicity of the vaccine in individuals who are chronically exposed to hookworm can be confidently extrapolated from studies performed in volunteers who have never been exposed to or infected with the parasite, since chronic infection is known to modulate the host’s immune response to this helminth (67). Provided that no significant safety concerns emerge during phase 1 testing of candidate hookworm vaccine antigens in hookworm-exposed adults, clinical testing will proceed into the pediatric population in which an eventual proof-ofprinciple study will be conducted; that is, in preschool and/or primary school-aged children. Young children are the target age-group for several reasons, but primarily because research has shown that although infections of high worm burden occur in both adults and children, the health impact of hookworm infection in terms of host blood loss resulting in anemia is greatest in children (28). In addition, future use of an effective HHV will likely be through integration into existing helminth control programs, which are currently based on the annual administration of anthelmintics to preschool and school-aged children (1,3). The general study design of a proof-of-principle phase 2 trial will entail assessment of the rate and intensity of

Chapter 75: Hookworm Vaccines

hookworm infection in vaccinated children, in comparison to those administered an active comparator vaccine. To properly assess any larval component of a hookworm vaccine, a critical element of the study design of a phase 2 trial will be the pretreatment of infected individuals with an anthelmintic, to eliminate any adult worms that are present in the vaccinees’ gastrointestinal tracts prior to vaccination. Estimation of efficacy will be made by measuring the vaccine’s impact on worm burden, which will be indirectly assessed by quantitative fecal egg counts. If a hookworm vaccine were to induce complete protection against new infections, an appropriate primary endpoint for an efficacy trial would therefore be the incidence of post-vaccination infection. However, a hookworm vaccine is likely to be non-sterilizing, but may nevertheless reduce the number of viable L3 that attain maturity; in this scenario, worm burden becomes a more appropriate efficacy endpoint. Measuring fecal egg counts is an appropriate indicator of the health impact of hookworm infection because of the demonstrated correlation between egg counts and host worm burden, as well as the relationship between egg counts and host blood loss as measured by quantifying fecal heme (28). Vaccination may also delay the onset of a new hookworm infection. In addition to fecal egg counts, fecal heme measurements and host hematological parameters will be important secondary endpoints for assessing the biologic activity of candidate hookworm vaccines in proof-of-principle studies (68). The goal of clinical development of the bivalent HHV is to demonstrate, in a proof-of-principle study, that the vaccine is effective in reducing the intensity of infection with hookworm and the clinical outcomes of hookworm disease (e.g., intestinal blood loss and IDA) in young children who are first exposed to hookworm or following anthelmintic treatment of hookworm-infected older children. Following this, the plan is for a middle-income country such as Brazil to continue the clinical development of the vaccine up to and including application for registration/licensure.

GLOBAL ACCESS OF THE HOOKWORM VACCINE The classification of hookworm infection as a neglected tropical disease presents great challenges for global access of the bivalent HHV. The term ‘‘global access’’ refers to the concept of rendering health solutions accessible to people who are most in need in developing countries. Among the major challenges that require consideration are those related to vaccine design, vaccine development and distribution, vaccine introduction, financing, knowledge dissemination, and management of intellectual property. HHVI-Sabin’s global access strategy has two underlying principles: (i) The HHV will be made available at affordable prices to those most in need in the developing world and (ii) Knowledge gained through discovery will be promptly made available to the broader scientific community.

Vaccine Design and Development Processes for the manufacture and formulation of the two larval and adult antigens and the bivalent HHV are being designed to maximize manufacturing yields and minimize costs, both essential elements for a product intended for the world’s poor. Production of these vaccines relies on the use of highyield expression systems (primarily yeast and/or bacterial),

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protein purification protocols that use low-cost column resins, and formulation with Alhydrogel. Additional manufacturing steps, including site-directed mutagenesis of genes are being explored to achieve or improve a thermostable vaccine that will not degrade in tropical and subtropical environments. For ease of conducting clinical trials in remote rural areas, the HHVI vaccines are formulated in single-dose vials for clinical trials, although ultimately multi-dose vials will be used for wide-scale delivery. As noted above, in the absence of a significant commercial market for the HHV, a major component of the HHVI-Sabin global access strategy is a commitment to partner with vaccine manufacturers and national health ministries in IDCs.

Vaccine Introduction and Financing The events surrounding the introduction of a new vaccine in developing countries represent a major challenge for global access. In the case of the HHV, the challenges for introduction include the sheer magnitude of the hookworm problem and its endemicity, primarily in rural areas, the need for international consensus on the use of hookworm vaccines, and the need for innovative health care delivery systems to distribute the vaccines. To further ensure timely vaccine introduction, HHVISabin has initiated an effort to establish international consensus on the use of the HHV. This is being done under the auspices of the Global Network for Neglected Tropical Disease Control, an alliance of the major neglected tropical disease public-private partnerships (11), and the WHO Department of Neglected Tropical Diseases. In 2005, the WHO called for the development of new control tools for hookworm, especially a vaccine (69). Central to obtaining international consensus is the concept that the HHV should be distributed in conjunction with schoolbased deworming programs that have already been mandated by a 2001 World Health Assembly resolution (69,70). Bergquist et al. (71) have coined the term ‘‘vaccine-linked chemotherapy’’ to describe the appropriate use of a vaccine following administration of an anthelminthic drug. Since 2001, however, preschool children between the ages of one and five years have emerged as an important target population for deworming, and global efforts at reaching this population through child health days have so far been more successful than reaching schoolaged children (69). Therefore, the HHVI is exploring administration of the HHV to very young and preschool children (and its administration through child health days) prior to their exposure to hookworms in the environment. Ultimately, the HHVI might be used in a pro-poor strategy that combines it with other low-cost interventions (11). Also critical to the global access of the HHV are the concepts of financing and cost effectiveness, which in turn are based on a number of parameters including transmission dynamics, disease burden, and health care delivery systems.

Intellectual Property and Knowledge Dissemination HHVI-Sabin has adopted a policy of regularly publishing its scientific findings in the peer-reviewed literature (preferably in open access journals) and in a timely manner. The HHVISabin’s decision to patent intellectual property is based on the assumption that doing so would support a global access strategy of partnering with selected IDC vaccine manufacturers, as patents are valuable tools to encourage investment.

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SUMMARY It is anticipated that from day one, the HHV will need to be made available for less than US$1 per dose, and possibly less. Among the challenges for HHVI-Sabin, the technology required to produce the HHV is at an equivalent level of sophistication to that required for more expensive recombinant vaccines such as for human papillomavirus, but through bypassing the large northern vaccine manufacturers and instead developing a recombinant vaccine through a nonprofit product development partnership in collaboration with an IDC vaccine manufacturer, the HHVI-Sabin plans to produce a recombinant product at a fraction of the usual cost. Equally important, the HHVI-Sabin is manufacturing a product that specifically targets the world’s poorest people rather than one that is intended primarily for North American and European markets and that then slowly trickles down to developing country populations over a period of decades. This new paradigm may be applied to a large of number of so-called antipoverty vaccines for neglected tropical diseases (14). With only three vaccines for neglected tropical diseases in clinical trials—hookworm infection, leishmaniasis, and schistosomiasis—we are clearly at the nascent stages of developing vaccines for neglected populations (14).

REFERENCES 1. Bethony J, Brooker S, Albonico M, et al. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 2006; 367:1521–1532. 2. De Silva Sliva NR, Brooker S, Hotez PJ, et al. Soil-transmitted helminth infections: updating the global picture. Trends Parasitol 2003; 19:547–551. 3. Hotez PJ, Brooker S, Bethony JM, et al. Hookworm infection. N Eng J Med 2004; 351:799–807. 4. Crompton DW. The public health importance of hookworm disease. Parasitology 2000; 121(suppl):S39–S50. 5. Christian P, Khatry SK, West KP. Antenatal anthelmintic treatment, birthweight, and infant survival in rural Nepal. Lancet 2004; 364:981–983. 6. Larocque R, Casapia M, Gotuzzo E, et al. A double-blind randomized controlled trial of antenatal mebendazole to reduce low birthweight in a hookworm-endemic area of Peru. Trop Med Int Health 2006; 11:1485–1495. 7. Chan MS. The global burden of intestinal nematode infections— fifty years on. Parasitol Today 1997; 13:438–443. 8. Miguel EA, Kremer M. Worms: identifying impacts on education and health in the presence of treatment externalities. Econometrica 2003; 72:159–217. 9. Bleakley H. Disease and development: evidence from hookworm eradication in the American South. Q J Econ 2007; 122:73–117. 10. Hotez PJ. Hookworm and poverty. Ann N Y Acad Sci 2008; 1136:38–44. 11. Hotez PJ, Molyneux DH, Fenwick A, et al. Control of neglected tropical diseases. N Engl J Med 2007; 357:1018–1027. 12. Hotez PJ, Bethony J, Bottazzi ME, et al. New technologies for the control of human hookworm infection. Trends Parasitol 2006; 22:327–331. 13. Diemert D, Bethony J, Hotez PJ. Hookworm vaccines. Clin Infect Dis 2008; 46:282–288. 14. Hotez PJ, Ferris M. The antipoverty vaccines. Vaccine 2006; 24: 5787–5799. 15. Brooker S, Bethony J, Hotez PJ. Human hookworm infection in the 21st century. Adv Parasitol 2004; 58:197–288. 16. Anderson RM, May RM. Helminth infections of humans: mathematical models, population dynamics, and control. Adv Parasitol 1985; 24:1–101.

17. Albonico M, Crompton DW, Savioli L. Control strategies for human intestinal nematode infections. Adv Parasitol 1999; 42: 277–341. 18. Bethony J, Chen J, Lin S, et al. Emerging patterns of hookworm infection influence of aging on the intensity of Necator infection in Hainan Province, People’s Republic of China. Clin Infect Dis 2002; 35:1336–1344. 19. Hotez PJ. China’s hookworms. China Q 2002; 172:1029–1041. 20. Humphries DL, Stephenson LS, Pearce EJ, et al. The use of human faeces for fertilizer is associated with increased intensity of hookworm infection in Vietnamese women. Trans R Soc Trop Med Hyg 1997; 91:518–520. 21. Brooker S, Alexander N, Geiger S, et al. Contrasting patterns in the small-scale heterogeneity of human helminth infections in urban and rural environments in Brazil. Int J Parasitol 2006; 36:1143–1151. 22. Quinnell RJ. Genetics of susceptibility to human helminth infection. Int J Parasitol 2003; 33:1219–1231. 23. Raso G, Luginbu¨hl A, Adjoua CA, et al. Multiple parasite infections and their relationship to self-reported morbidity in a community of rural Coˆte d’Ivoire. Int J Epidemiol 2004; 33:1092–1102. 24. Fleming FM, Brooker S, Geiger SM, et al. Synergistic associations between hookworm and other helminth species in a rural community in Brazil. Trop Med Int Health 2006; 11:56–64. 25. Brooker S, Beasley M, Ndinaromtan M, et al. Use of remote sensing and geographical information system in a national helminth control programme in Chad. Bull World Health Organ 2002; 80:783–789. 26. Appleton CC, Gouws E. The distribution of common intestinal nematodes along an altitudinal transect in KwaZulu-Natal, South Africa. Ann Trop Med Parasitol 1996; 90:181–188. 27. Mabaso ML, Appleton CC, Hughes JC, et al. The effect of soil type and climate on hookworm (Necator americanus) distribution in KwaZulu-Natal, South Africa. Trop Med Int Health 2003; 8:722–727. 28. Stoltzfus RJ, Dreyfuss ML, Chwaya HM, et al. Hookworm control as a strategy to prevent iron deficiency. Nutr Rev 1997; 55:223–232. 29. Brooker S, Jardim-Botelho A, Quinnell RJ, et al. Age-related changes in hookworm infection, anaemia and iron deficiency in an area of high Necator americanus hookworm transmission in south-eastern Brazil. Trans R Soc Trop Med Hyg 2007; 101:146–154. 30. Brooker S, Hotez PJ, Bundy DAP. Hookworm-related anaemia among pregnant women: a systematic review. PLoS Negl Trop Dis 2008; 2(9):e291. 31. Bundy DA, Chan MS, Savioli L. Hookworm infection in pregnancy. Trans R Soc Trop Med Hyg 1995; 89:521–522. 32. Hotez PJ, Molyneux DH, Fenwick A, et al. Incorporating a rapidimpact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med 2006; 3:e102. 33. Quinnell RJ, Pritchard DI, Raiko A, et al. Immune responses in human necatoriasis: association between interleukin-5 responses and resistance to reinfection. J Infect Dis 2004; 190:430–438. 34. Loukas A, Constant SL, Bethony JM. Immunobiology of hookworm infection. FEMS Immunol Med Microbiol 2005; 43:115–124. 35. Fujiwara RT, Geiger SM, Bethony J, et al. Comparative immunology of human and animal models of hookworm infection. Parasite Immunol 2006; 28:285–293. 36. Quinnell RJ, Bethony J, Pritchard DI. The immunoepidemiology of human hookworm infection. Parasite Immunol 2004; 26:443–454. 37. Mendez S, Valenzuela J, Wu WH, et al. Host cytokine production, lymphoprolieration, and antibody responses during the course of Ancylsotoma cenylanicum infection in the golden syrian hamster. Infect Immun 2005; 73:3402–3407. 38. Xue J, Qian HQ, Yao J, et al. Nector americanus: optimization of the golden hamster model for testing anthelminthic drugs. Exp Parasitol 2005; 111:219–223. 39. Xiao S, Zhan B, Xue J, et al. The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus. Exp Parasitol 2008; 118:32–40.

Chapter 75: Hookworm Vaccines 40. Pit DS, Polderman AM, Baeta S, et al. Parasite-specific antibody and cellular immune responses in humans infected with Necator americanus and Oesophagostomum bifurcum. Parasitol Res 2001; 87:722–729. 41. Geiger SM, Massara CL, Bethony J, et al. Cellular responses and cytokine production in posttreatment hookworm patients from an endemic area in Brazil. Clin Exp Immunol 2004; 136:334–340. 42. Geiger SM, Caldas IR, Mc Glone BE, et al. Stage-specific immune responses in human necator americanus infection. Parasite Immunol 2007; 29:347–358. 43. Fujiwara RT, Loukas A, Mendez S, et al. Vaccination with irradiated Ancylostoma caninum third stage larvae induces a Th2 protective response in dogs. Vaccine 2005; 24:501–509. 44. Fujiwara RT, Zhan B, Mendez S, et al. Protection against hookworm infection elicited by vaccination with recombinant Ac-16 is mediated by reduction of worm fecundity and canine host blood loss. Clin Vaccine Immunol 2007; 14:281–287. 45. Bethony J, Loukas A, Smout M, et al. Antibodies against a secreted protein from hookworm larvae reduce the intensity of hookworm infection in humans and vaccinated laboratory animals. FASEB J 2005; 19:1743–1745. 46. Loukas A, Prociv P. Immune responses to hookworm infections. Clin Microbiol Rev 2001; 14:689–703. 47. Druilhe P, Tall A, Sokhna C. Worms can worsen malaria: towards a new means to roll back malaria? Trends Parasitol 2005; 21:359–362. 48. Mwangi TW, Bethony JM, Brooker S. Malaria and helminth interactions in humans: an epidemiological viewpoint. Ann Trop Med Parasitol 2006; 100:551–570. 49. Ghosh K, Wu W, Antoine AD, et al. The impact of concurrent and treated Ancylostoma ceylancium hookworm infections on the immunogenicity of a recombinant hookworm vaccine in hamsters. J Infect Dis 2006; 193:155–162. 50. Hotez PJ, Zhan B, Bethony JM, et al. Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative. Int J Parasitol 2003; 33:1245–1258. 51. Hotez PJ, Hawdon JM, Cappello M, et al. Molecular approaches to vaccinating against hookworm disease. Pediatr Res 1996; 40:515–521. 52. Loukas A, Bethony J, Brooker S, et al. Hookworm vaccines: past, present, and future. Lancet Infect Dis 2006; 6:733–741. 53. Miller TA. Vaccination against the canine hookworm diseases. Adv Parasitol 1971; 9:155–183. 54. Miller TA. Industrial development and field use of the canine hookworm vaccine. Adv Parasitol 1978; 16:333–342. 55. Hotez PJ, Ghosh K, Hawdon JM, et al. Experimental approaches to the development of a recombinant hookworm vaccine. Immunol Rev 1999; 171:163–171. 56. Asojo OA, Goud G, Dhar K, et al. X-ray structure of Na-ASP-2, a pathogenesis-related-1 protein from the nematode parasite, Necator americanus, and a vaccine antigen for human hookworm infection. J Mol Biol 2005; 346:801–814.

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57. Goud GN, Bottazzi ME, Zhan B, et al. Expression of the Necator americanus hookworm larval antigen Na-ASP-2 in Pichia pastoris and purification of the recombinant protein for use in human clinical trials. Vaccine 2005; 23:4754–4764. 58. Mendez S, Zhan B, Goud G, et al. Effect of combining the larval antigens Ancylostoma secreted protein 2 (ASP-2) and metalloprotease 1 (MTP-1) in protecting hamsters against hookworm infection and disease caused by Ancylostoma ceylanicum. Vaccine 2005; 23:23–30. 59. Goud GN, Zhan B, Ghosh K, et al. Cloning, yeast expression, isolation and vaccine testing of recombinant ancylostoma secreted protein 1 (ASP-1) and ASP-2 from Ancylostoma ceylanicum. J Infect Dis 2004; 189:919–929. 60. Loukas A, Bethony JM, Williamson AL, et al. Vaccination of dogs with a recombinant cysteine protease from the intestine of canine hookworms diminishes fecundity and growth of worms. J Infect Dis 2004; 189:1952–1961. 61. Loukas A, Bethony JM, Mendez S, et al. Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs. PLoS Med 2005; 2:e295. 62. Zhan B, Liu S, Perally S, et al. Vaccine potential of a heme binding glutathione transferase (GST) from adult hookworm Ancylostoma caninum. Infect Immun 2005; 73:6903–6911. 63. Asojo OA, Homaa K, Sedlacek M, et al. X-ray structures of NaGST-1 and NaGST-2 two glutathione S-transferase from the human hookworm Necator americanus. BMC Struct Biol 2007; 7:42. 64. Bethony JM, Simon G, Diemert D, et al. Randomized, placebocontrolled, double-blind trial of the Na-ASP-2 hookworm vaccine in unexposed adults. Vaccine 2008; 26:2408–2417. 65. Morel CM, Acharya T, Broun D, et al. Health innovation: the neglected capacity of developing countries to address neglected diseases. Science 2005; 309:401–404. 66. Bottazzi ME, Miles AP, Diemert D, et al. An ounce of prevention on a budget: a nonprofit approach to developing vaccines against neglected diseases. Expert Rev Vaccines 2006; 5:189–198. 67. Maizels RM, Balic A. Resistance to helminth infection: the case for interleukin-5-dependent mechanisms. J Infect Dis 2004; 190: 427–429. 68. Brooker S, Bethony JM, Rodrigues LC, et al. Epidemiologic, immunologic and practical considerations in developing and evaluating a human hookworm vaccine. Expert Rev Vaccines 2005; 4:35–50. 69. World Health Organization. Deworming for Health and Development 2005; report of the third global meeting of the partners for parasite control, Geneva 29 to 30, November 2004. 70. World Bank. School deworming at a glance.; Washington D.C., World Bank, 2003. Available at: http://siteresources.worldbank. org/INTPHAAG/Resources/AAGDewormingEng110603.pdf. 71. Bergquist NR, Leonardo LR, Mitchell GF. Vaccine-linked chemotherapy: can schistosomiasis control benefit from an integrated approach? Trends Parasitol 2005; 21:112–117.

76 Improved Smallpox Vaccines Matthew E. Cohen and Stuart N. Isaacs Department of Medicine, Division of Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.

INTRODUCTION The first preventative vaccines against an infectious agent were orthopoxviruses, which were used to prevent smallpox (1). Vaccination with live vaccinia virus resulted in cross-protection against infection by variola virus (VARV), the causative agent of the deadly disease smallpox. Vaccination with vaccinia virus was part of the World Health Organization’s (WHO) successful strategy to eradicate smallpox, which made smallpox the first, and currently only, disease to be eradicated from plaguing humankind (1). However, this success has now led to concern about the accidental or intentional release of VARV (2,3). Because routine vaccination of civilians with vaccinia virus vaccines ended with the eradication of smallpox, a large portion of the world population is susceptible, and for those previously vaccinated, full protection from smallpox may be incomplete. In addition to concerns about VARV, ongoing outbreaks of monkeypox in Africa (4,5) and the potential spread of monkeypox to nonendemic areas (6), has generated the necessity to have smallpox vaccine available. However, concerns about the safety of the smallpox vaccines used in the smallpox eradication program have led to the development of new orthopoxvirus vaccines (7–10).

LIVE VACCINIA VIRUS–BASED VACCINES Development of a Vaccine Propagated in Cell Culture The smallpox vaccines that were used around the world to combat smallpox were developed in a time well before modern methods of growing and passaging live virus vaccines. In the United States, the smallpox vaccine used was Dryvax (manufactured by Wyeth Laboratories, Marietta, Pennsylvania, U.S.), and it was grown and passaged in the skin of animals. Other parts of the world used other strains of vaccinia virus, and depending upon the strain, different rates of vaccine-related severe complications (e.g., death or post-vaccination encephalitis) were observed (11). With the eradication of naturally occurring infections from VARV, routine smallpox vaccination ended because the risks (both major and minor) of the vaccine were deemed to be too high in the setting of no disease. Without widespread use of the vaccine, production ended. While the Centers for Disease Control and Prevention (CDC) had access to old stocks of Dryvax, there were not enough doses available to combat a large-scale outbreak. Thus, there was great urgency to produce and stockpile more vaccine using modern vaccine-manufacturing procedures. However, a major dilemma was that the virus

contained in the Dryvax vaccine represented a swarm of virus with unlimited diversity (Russell Regnery, personal communication). A number of individual virus plaques from stocks of Dryvax were picked and characterized. The virus propagated from a single plaque that most closely resembled the in vitro and in vivo characteristics of the Dryvax swarm (but resulted in lower neurotoxicity in mice) was selected (12). This virus, grown in cell culture, was shown to protect animals against orthopoxvirus infections (12–14). Ultimately, this virus, called ACAM2000, became the FDA-licensed smallpox vaccine in the United States (15,16). However, like Dryvax, this vaccine generates infectious progeny that can spread within the host, and thus it is expected to result in the same minor and major complications of the parent virus (17,18). Therefore, the prescribing package insert for ACAM2000 contains the same warnings as Dryvax indicating that people with immunodeficiencies, common skin disorders like eczema and atopic dermatitis, cardiac disease, age less than 12 months, and pregnancy are still at risk for developing a severe complication from the vaccine, and should not get vaccinated in the setting where there is no active smallpox (19). Because of the concerns about complications from the smallpox vaccine, there has been a continued interest in developing safer smallpox vaccines that can be used in a diverse population, including people at risk for the severe complications from current live vaccinia virus vaccines.

Development of More Attenuated Live Virus Vaccines Prior to the eradication of smallpox, some countries began testing more attenuated strains of vaccinia virus as potential smallpox vaccines. These vaccines were isolated by continual passage of a parental vaccine strain in cell culture, which resulted in random mutations that attenuated the virus. Many of these vaccines were given to large populations and appeared to have less side effects, but were used in countries where smallpox disease no longer existed. Examples of this strategy are LC16m8 and MVA strains of vaccinia virus. LC16m8 In Japan, LC16m8 was developed as an attenuated vaccinia virus vaccine (20–22). This virus was isolated after passage of the parental Lister strain of vaccinia virus in rabbit cells at low temperature. The resulting virus made very small plaques in cell culture and showed less neurovirulence in animal models. Much of the attenuated phenotype of this virus is due to a mutation in the B5R gene (23), which encodes for an important

Chapter 76: Improved Smallpox Vaccines

glycoprotein needed for the optimal production of an infectious form of virus critical for virus spread. Thus, while LC16m8 can grow and make infectious particles, it spreads poorly in cell culture. LC16m8 has been shown to generate protective immunity in mice (24), rabbits (24), and nonhuman primates (25). However, there are two important concerns about this vaccine. Since the key attenuating mutation in B5R is a one-base deletion that results in a frame-shift and early truncation of the B5 protein (23), there is evidence that virus can revert to wild-type during growth (26). An additional concern is that the B5 protein is an important protective target of the humoral immune response to live vaccinia virus vaccination (27,28), and this aspect of protection may be lost in an LC16m8 vaccinated individual. Modified Vaccinia Ankara In Germany, modified vaccinia Ankara (MVA) was developed as a highly attenuated potential smallpox vaccine (29,30). This vaccine was obtained after 572 serial passages of the parental vaccinia strain on chick embryo fibroblasts. This resulted in about 25 kilobases deleted and a virus that no longer produced infectious progeny virus in almost all mammalian cell lines. That is, the virus could infect, replicate its DNA, and generate abundant amounts of key viral proteins, but could not assemble into infectious virions in most mammalian cells. Because of the inability to generate infectious virions in human cells, this type of virus would likely be safe to give to many people who have conditions that would not allow routine smallpox vaccination. Therefore, this virus has been intensively studied as a next generation smallpox vaccine that may ultimately gain FDA approval in the United States. The virus has been widely studied and shown to generate antibody responses similar to Dryvax (31), as well as protection in mouse (32,33) and nonhuman primate challenge models (34–36). There is evidence that MVA vaccination results in more rapid protection when compared to a fully replication competent vaccine, like Dryvax (37). While the mechanism for this enhanced early protection by MVA is not entirely known, part of the explanation may be that it induces more rapid immunity (37) because it is given at about a 1000 times higher dose than current replication competent vaccinia vaccines. It also appears that MVA can activate innate immune responses because it is missing genes present in replication competent vaccinia virus that encode proteins that may initially dampen the immune response (38,39).

SUBUNIT-BASED VACCINES Until recently, it was believed that protection conferred by live vaccinia virus vaccination was predominantly due to antivaccinia T-cell responses. This was mainly based on the fact that inactivated smallpox vaccines did not protect against smallpox (1). Thus, it was assumed that live vaccinia virus vaccination protected by potent antiviral T-cell responses. However, the inability of experimental inactivated vaccines to protect may have been due to denaturing of key targets (40) as well as the fact that the vaccine preparations did not contain some critical antigens that are present on a minor population of infectious virus (41,42). Furthermore, in recent years, protection via vaccination with live vaccinia virus has been shown to be dependent on vaccinia specific, CD4 þ dependent B-cell responses (43–47). Thus, future-generation smallpox vaccines that are capable of inducing protective antibody responses are viable alternatives to the current live-virus vaccines. One way

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to induce such antibody responses is to provide protein(s) directly to the immune system to which neutralizing and protective antibodies can be generated. Strategies to present these critical proteins include direct injection of soluble proteins with adjuvants, introduction of recombinant DNA that host cells transcribe and translate, and live or attenuated vectors that deliver poxvirus proteins to the host immune system. Since poxviruses are large DNA viruses that encode over 200 proteins, the identification of suitable proteins that would generate a protective immune response is complex. Most research has focused on different surface membrane proteins of the two infectious forms of virus, the mature virus (MV) and the extracellular virus (EV) (48,49). Furthermore, including targets against both MV and EV appears to provide the best protection from morbidity and mortality (50–54). Including targets against both forms of infectious virus is believed to provide a way to decrease the infecting inoculum (believed to be mainly MV), and then alter the spread and dissemination of the virus within an infected host (thought to be mainly EV) (55–59). Initial insights into appropriate targets against MV and/or EV proteins were based on the production of antibodies that could neutralize virus in vitro or provide passive protection against vaccinia virus challenge in vivo (50,60–71). Relevant protein targets were also identified by examining what proteins were recognized by vaccinia immunoglobulin (VIG) (27,28,72–74), serum from vaccinia virus vaccinated individuals that was used clinically to treat complications from live vaccinia virus vaccination. Many of the protein targets identified by these approaches are targets of potent neutralizing antibodies. The following sections and tables will cover the most widely studied viral targets and the effort that is being made to combine these targets into effective subunit vaccines.

Protein-Based Subunit Vaccines The first successful attempt at a subunit vaccine to protect against lethal vaccinia virus challenge was by Lai, et al. in 1991 (75). They intraperitoneally injected purified vaccinia virus A27 protein (an MV protein) generated in Escherichia coli and found that the antibody response generated was both MV neutralizing in vitro and 100% protective against a lethal intraperitoneal challenge with vaccinia virus. The EV proteins A33 and B5, produced in baculovirus, were first shown to generate protective immune responses by Galmiche et al. (65). They found that injection of A33 or B5 protein provided 100% protection from lethal intranasal challenge with vaccinia virus. While only B5 vaccination elicited in vitro EV neutralizing activity, the antibodies produced against A33 resulted in ‘‘comet inhibition,’’ indicating that they altered the way EV spread in cell culture. Antibody to A33 may also provide protection through the activation of complement (76). Table 1 summarizes the individual orthopoxvirus genes that have been examined as a subunit vacc ine. Pro teins hav e been express ed i n bact eria (44,47,65,75,77–79), baculovirus (52,53,65,80), and even recombinant plants (81). While work with individual proteins has helped identify appropriate targets to include in a subunit vaccine, the combination of multiple proteins is believed to provide the optimum protection (Table 2). For example, A33, B5, and L1 proteins have been used in combination to generate a mouse antibody response to both the MV and EV infectious forms of vaccinia virus (52,53). These trivalent subunit vaccines provide 100%

A33R (EV)

A27L (MV)

A13L (MV)

Gene(s) (target)

DNA, IM, 4 doses

DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in baculovirus system), SC, 4 doses DNA, IM, 4 doses

VACV, IHD-J strain

VACV, NYBHCONN strain

Protein (expressed in bacteria), SC, 2 doses VRP, 2 doses of 1E6 IU

Replication incompetent rAd35 vector, 1 dose

VACV, IHD-J strain ECTV (EVM135)

CPXV

VACV, WR strain

VACV, WR strain

Protein (expressed in baculovirus system), SC, 4 doses

DNA, gene gun (abdominal epidermis), 4 doses Replication incompetent rAd35 vector, 1 dose

VEE virus replicon (VRP), 2 doses of 1E6 IU VRP

DNA, gene gun (abdominal epidermis), 3 doses DNA, IM, 4 doses

Protein (expressed in bacteria), IP, 2 doses

DNA, IM, 4 doses

Delivery

VACV, IHD-J strain

VACV, WR strain

VARV

VACV, IHD-J strain CPXV

VACV, NYBHCONN strain

VACV, IHD-J strain VACV, WR strain

Origin

Table 1 Individual Gene Targets

None

None

Freund’s incomplete

BALB/c mice

BALB/c mice

BALB/c mice BALB/c mice

BALB/c mice

Ribi or QS21

None

BALB/c mice

BALB/c mice

BALB/c mice

BALB/c mice

BALB/c

CPXV, Brighton red/ IN/1E6 pfu/mo (sublethal dose) VACV, WR strain/IP/ 2E8 pfu/mo

VACV, WR strain/IN/ 1E6 pfu/mo or 2E7 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo ECTV/footpad/ 3000pfu/mo

VACV, WR/IP/5E8 pfu/mo

VACV, IHD-J strain/ IN/1E6 pfu/mo

VACV, IHD-J strain/ IN/1E7 pfu/mo

VACV, WR strain/IP/ 2E8 pfu/mo

VACV, IHD-J strain/ IN/1E7 pfu/mo CPXV, Brighton red/ IN/1E6 pfu/mo (sublethal dose) VACV, WR strain/IP/ 5E7 pfu/mo

VACV, WR/IP/5E8 pfu/mo

BALB/c mice BALB/c mice BALB/c mice

VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, WR strain/IP/ 1.5E8 pfu/mo

Challenge virus/ route/dose

BALB/c mice BALB/c mice

Animal model

DNA precipitated on gold

Primary: Freund’s complete/Boosts: Freund’s incomplete None

None

None

None

None

Prime: Freund’s complete/Boosts: Freund’s incomplete DNA precipitated on gold

None

Adjuvant

75%/moderate to severe

100%/moderate

70%/moderate

67%/significant

Anti-VACV antibody, not comet inhibitory, Th1/Th2 balanced response (IgG2a and IgG1 both produced), IFNg producers detected

Anti-VACV antibody, comet inhibitory, antigen specific CD8þ T cells Anti-VACV antibody, Th1 response (IgG2a dominant)

Anti-VACV antibody, Th2 response (IgG1 dominant), comet inhibitory Anti-VACV antibody

1E6: 100%/mild 2E7: 70%/severe

0%/severe

Anti-VACV antibody, protection not correlated with titers Anti-VACV antibody

Anti-VACV antibody, not comet inhibitory, Th1/Th2 balanced response (IgG2a and IgG1 both produced), IFNg producers detected Anti-VACV antibody

MV NAb

Anti-VACV antibody, Th1 response (IgG2a dominant)

No antibody detected

MV NAb

MV NAb

No antibody detected

100%/significant

100%/significant

50%/severe

100%/moderate

100%/significant

80%/significant

10%/severe

100%/ND

0%/severe

Correlates of protection Survival/morbidity investigated Reference

54

92

47

88

52

50

65

65

54

79

92

88

51

75

88

840 Cohen and Isaacs

B5R (EV)

A56R (EV)

A36R (Cell*a)

A34R (EV)

Gene(s) (target)

CPXV, de novo synthesis

VACV, WR strain

VARV

VACV, IHD-J strain VACV, WR strain CPXV

VACV, WR strain

VACV, IHD-J strain VACV, NYBHCONN strain

VACV, IHD-J strain VACV, IHD-J strain VACV, IHD-J strain VACV, IHD-J strain

Replication incompetent rAd5 vector, IM, 1 dose

DNA, gene gun (abdominal epidermis), 4 doses Replication incompetent rAd35 vector, IM, 1 dose

Protein (expressed in planta), IM, 3 doses VRP, 2 doses of 1E6 IU

BALB/c mice

BALB/c mice

None

None

BALB/c mice

BALB/c mice BALB/c mice BALB/c mice

BALB/c mice

BALB/c mice BALB/c mice

BALB/c mice BALB/c mice BALB/c mice BALB/c mice

3 months post vaccination: VACV, WR strain/ IN/1E5 pfu/mo (sublethal)

VACV, WR strain/IP or IN/IP: 2E8 pfu/ mo, IN: 2E7 pfu/ mo

VACV, WR strain/IN/ 1E6 pfu/mo or 2E7 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, WR strain/IN/ 1.2E6 pfu/mo CPXV, Brighton red/ IN/1E6 pfu/mo (sublethal) VACV, WR strain/IP/ 5E7 pfu/mo

VACV, IHD-J strain/ IN/1E6 pfu/mo VACV, WR/IP/5E8 pfu/mo

VACV, IHD-J strain/ IN/1E6 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo

VACV, IHD-J strain/ IN/1E6 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, IHD-J strain/ IN/1E7 pfu/mo

VACV, IHD-J strain/ IN/1E7 pfu/mo

BALB/c mice

BALB/c mice BALB/c mice BALB/c mice

Challenge virus/ route/dose

Animal model

None

None

CpG, alum

None

Ribi or QS21

DNA precipitated on gold

DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in baculovirus system), SC, 4 doses DNA, IM, 3 or 4 doses

DNA, IM, 4 doses

Primary: Friend’s complete/Boosts: Friend’s incomplete None

Protein (expressed in baculovirus system), SC, 4 doses

DNA, IM, 4 doses

None

None

DNA, IM, 4 doses DNA, IM, 4 doses

DNA, IM, 4 doses Primary: Friend’s complete/Boosts: Friend’s incomplete None

None

DNA, IM, 4 doses

VACV, IHD-J strain VACV, IHD-J strain VACV, IHD-J strain Protein (expressed in bacteria), SC, 4 doses

Primary: Friend’s complete/Boosts: Friend’s incomplete None

Adjuvant

Protein (expressed in bacteria), SC, 4 doses

Delivery

VACV, IHD-J strain

Origin

Table 1 (Continued )

NR

*50%/very severe 60%/significant

Anti-VACV antibody, comet inhibitory, Th1/Th2 balanced response (IgG2a and IgG1 both produced), minimal IFNg producers detected Anti-VACV antibody, comet inhibitory

IP: *90%/severe IN: 75%/severe

100%/moderate

Anti-VACV antibody

100%/significant

100%/significant

100%/severe

93

54

79

92

81

88

(continued )

Anti-VACV antibody, IFNg Type I response Anti-VACV antibody, comet inhibitory Anti-VACV antibody, Th1 response (IgG2a dominant)

52

Anti-VACV antibody, Th2 response (IgG1 dominant) 1E6: 100%/moderate 2E7: 30%/severe 100%/moderate

65 51

NR

*80%/significant

65

Anti-VACV antibody, non-MV NAb

Anti-VACV antibody

*90%/severe

88

88

65

65

88

65

65

Reference

40%/severe

Anti-VACV antibody

50%/significant

No antibody detected

NR

0%/severe

No antibody detected

NR

*20%/severe 0%/severe

NR

0%/severe

Correlates of protection Survival/morbidity investigated

Chapter 76: Improved Smallpox Vaccines 841

VACV, NYBHCONN strain

L1R (MV)

NHP

Ribi or QS21

DNA precipitated on gold

Replication incompetent rAd35 vector, 1 dose

BALB/c mice

DNA precipitated on gold

DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in baculovirus system), SC, 4 doses DNA, gene gun (abdominal epidermis), 4 doses DNA, IM, 4 doses None

BALB/c mice BALB/c mice

BALB/c mice

Ribi

Protein (expressed in bacteria), IM, 2 doses

None

BALB/c mice BALB/c mice

None

BALB/c mice

BALB/c mice BALB/c mice

BALB/c mice

BALB/c mice

BALB/c mice

Animal model

VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, WR strain/IP or IN/IP: 2E8 pfu/ mo, IN: 2E7 pfu/ mo

VACV, WR strain/IN/ 1E6 pfu/mo or 2E7 pfu/mo MPXV/IV/2E7 pfu per NHP

VACV, WR/IP/5E8 pfu/mo

VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, WR strain/IN/ *5E5 pfu/mo or *2.5E7 pfu/mo

VACV, WR strain/IP/ 5E7 pfu/mo

VACV, IHD-J strain/ IN/1E7 pfu/mo VACV, WR strain/IP/ 5E7

3 months post vaccination: VACV, WR strain/ IN/1E5 pfu/mo (sublethal) 3 months post vaccination: VACV, WR strain/ IN/1E5 pfu/mo (sublethal) ECTV/Footpad/ 300pfu/mo(60xLD)

Challenge virus/ route/dose

No antibody detected MV NAb, not comet inhibitory, Th1/Th2 balanced response (IgG2a and IgG1 both produced), IFNg producers detected

IP: 100%/mild IN: 75%/severe

MV NAb

54

88

91

52

MV NAb, Th2 response (IgG1 dominant)

0%/severe

1E6: 100%/moderate 2E7: 30%/severe 100%/severe

77

50

MV NAb

*5E5:100%/significant *2.5E7:0%/ severe 89%/significant

88

79

89

88

83

93

93

Reference

MV NAb

No antibody detected

MV NAb

MV NAb

Anti-VACV antibody

Anti-ECTV antibody, neutralized protein activity

Anti-VACV antibody, comet inhibitory

Anti-VACV antibody, slightly comet inhibitory

33%/severe

100%/moderate

100%/moderate

50%/severe

100%/mild to moderate

100%/mild

53%/significant

Correlates of protection Survival/morbidity investigated

a

Cell*, A36 is a viral protein expressed on an infected cell that helps direct MV to become EV. Abbreviations: alum, aluminum hydroxide; CPXV, cowpox virus (Brighton red strain); ECTV, ectromelia virus (Moscow strain); IHD-J, International Health Department strain J of VACV; ID, intradermal; IM, intramuscular; IP, intraperitoneal; IRM, immune response modifiers; IV, intravenous; MPXV, monkeypox (Zaire 79 strain); MV, mature virus; NAb, neutralizing antibody; ND, not done; NHP, nonhuman primate; NR, not reported; NYBH-CONN, New York Board of Health-Connaught strain; pfu/mo, plaque-forming unit per mouse; QS21, nontoxic saponin derived from the soapbark tree, Quillaja saponaria; rAd, recombinant adenovirus; Ribi, MPLþTDM (monophosphoryl lipid A þ trehalose dicorynomycolate) adjuvant; rVSV, recombinant Vesicular stomatitis virus; SC, subcutaneous; IN, intranasal; VACV, vaccinia virus; VARV, variola virus (India 1967 strain); VRP, Venezuelan equine encephalitis (VEE) virus replicon particle; WR, western reserve strain of vaccinia virus; IFNg, interferon gamma.

VACV, IHD-J strain VACV, WR strain

VACV, NYBHCONN strain

VACV, WR strain

VACV, IHD-J strain VACV, WR strain

None

None

DNA, gene gun (abdominal epidermis), 4 doses DNA, gene gun (abdominal epidermis), 4 doses DNA, IM, 4 doses

VARV

None

DNA, IM, 4 doses

None

None

VACV, IHD-J strain VACV, WR strain

Replication incompetent rAd5 vector prime, rVSV boost, IN, 1 dose each

CPXV, de novo synthesis

None

Protein (expressed in bacteria), IM, 3 doses

rVSV, IM, 1 dose

CPXV, de novo synthesis

Adjuvant

ECTV

Delivery

Origin

H3L (MV)

B18R/ EVM166 (IRM) D8L (MV)

Gene(s) (target)

Table 1 Individual Gene Targets (Continued )

842 Cohen and Isaacs

VACV, NYBHCONN strain

VACV, NYBHCONN strain

A27L, A33R

A27L, B5R

B5R, L1R

A33R, L1R

VACV, NYBHCONN strain

A33R, B5R

VACV, WR strain

VACV, NYBHCONN strain

VACV, WR strain

VACV, WR strain

VACV, NYBHCONN strain

VACV, WR strain

VACV, NYBHCONN strain

A27L, L1R

VACV, WR strain

Origin

Genes

DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in baculovirus system), SC, 4 doses

DNA, gene gun (abdominal epidermis), 3 doses DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in baculovirus system), SC, 4 doses DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in baculovirus system), SC, 4 doses Protein (expressed in baculovirus system), SC, 2 doses

DNA, gene gun (abdominal epidermis), 3 doses DNA, gene gun (abdominal epidermis), 3 doses Protein (expressed in bacteria), SC, 3 doses

Delivery

Table 2 Combination Gene Targets

BALB/c mice

DNA precipitated on gold

Ribi or QS21

BALB/c mice

BALB/c mice

BALB/c mice

alum, or alum þ CpG, or Ribi, or QS21

DNA precipitated on gold

BALB/c mice

BALB/c mice

Ribi or QS21

DNA precipitated on gold

BALB/c mice

BALB/c mice

DNA precipitated on gold

Ribi or QS21

BALB/c mice

BALB/c mice

BALB/c mice

Animal model

Ribi or TiterMax gold adjuvant

DNA precipitated on gold

DNA precipitated on gold

Adjuvant

VACV, WR strain/IN/ 2E7 pfu/mo

VACV, IHD-J strain/ IP/5E8 pfu/mo

VACV, WR strain/IN/ 2E7 pfu/mo

VACV, WR strain/IN/ 2E7 pfu/mo

VACV, WR/IP/5E8 pfu/mo

VACV, WR strain/IN/ 2E7 pfu/mo

VACV, IHD-J strain/ IP/5E8 pfu/mo

VACV, IHD-J strain/ IP/5E8 pfu/mo

VACV, WR strain/IN/ *2.4E6 pfu/mo

VACV, IHD-J strain/ IP/5E8 pfu/mo

VACV, IHD-J strain/ IP/5E8 pfu/mo

Challenge virus/ route/dose

MV NAb, Th2 response (IgG1 dominant), comet inhibitory

Anti-VACV antibody, Th2 response (IgG1 dominant), comet inhibitory MV NAb

Anti-VACV antibody

MV NAb, comet inhibitory, Th1/Th2 balanced response in Ribi (equal IgG1 and IgG2a, Th2 response in TiterMax adjuvant (IgG1 dominant), Cd8þ and CD4þ responses MV NAb

MV NAb

Anti-VACV antibody

60%/severe

52

51

80

52

50

52

51

51

78

51

51

Reference

(continued )

MV NAb, Th2 response (IgG1 dominant)

alum: 50%/severe, MV Nab (except alum—some alum þ CpG: anti-VACV antibody), 100%/ comet inhibitory, Th1/Th2 moderate, Ribi: balanced response in alum 80%/signifþ CpG or Ribi (except for icant, QS21: anti-L1) or QS21 (equal 100%/mild IgG1 and IgG2a), Th2 response in alum (IgG1 dominant) 100%/moderate Weakly MV NAb

90%/moderate

100%/mild

90%/severe

80%/severe

40%/severe

100%/significant

95%/significant

70%/severe

Correlates of protection Survival/morbidity investigated

Chapter 76: Improved Smallpox Vaccines 843

A27L, A33R, B5R, L1R

A33L, B5R, L1R

A27L, B5R, D8L

VACV, WR strain

A27L, A33R, B5R

VACV, NYBHCONN strain

VACV, NYBHCONN strain

VACV, WR strain

VACV, WR strain

VACV, WR strain

VARV

DNA, gene gun (abdominal epidermis), 3 doses DNA, gene gun (abdominal epidermis), 4 doses

Protein (expressed in baculovirus system), IM, 2 doses

Protein (expressed in bacteria), 2 doses DNA, gene gun (abdominal epidermis), 4 doses DNA, gene gun (abdominal epidermis), 2 doses Protein (expressed in baculovirus system), SC, 3 or 4 doses Protein (expressed in baculovirus system), IM, 4 doses

VARV

VARV

Protein (expressed in bacteria), SC, 3 doses

Protein (expressed in baculovirus system), IM, 2 doses VRP, 2 doses of 1E6 IU

Delivery

VACV, WR strain

CPXV

Origin

Genes

Table 2 Combination Gene Targets (Continued )

NHP

DNA precipitated on gold

BALB/c mice

CpG, alum

BALB/c mice

NHP

QS21

DNA precipitated on gold

BALB/c mice

BALB/c mice

BALB/c mice BALB/c mice

BALB/c mice

BALB/c mice

BALB/c mice

Animal model

Ribi or QS21

none

none

Freund’s incomplete

Ribi or TiterMax gold adjuvant

none

CpG, alum

Adjuvant

MPXV/IV/2E7/NHP

VACV, WR strain/IN/ *6E6 pfu/mo OR ECTV/IN/1000 pfu/mo VACV, IHD-J strain/ IP/5E8 pfu/mo

MPXV/IV/5E7 (sublethal dose in this study)

VACV, WR strain/IN/ 2E7 pfu/mo

VACV, WR strain/IN/ 5E6 pfu/mo

VACV, WR strain/IN/ 5E6 pfu/mo VACV, WR strain/IP/ 5E7 pfu/mo

CPXV, Brighton red/ IN/1E6 pfu/mo (sublethal dose) VACV, WR strain/IN/ *2.4E6 pfu/mo

VACV, WR strain/IN/ *6E6 pfu/mo

Challenge virus/ route/dose

MV NAb, Th2 response (IgG1 dominant), comet inhibitory

MV NAb

MV NAb, comet inhibitory, Th1/Th2 balanced response in Ribi (equal IgG1 and IgG2a), Th2 response in TiterMax adjuvant (IgG1 dominant), Cd8þ and CD4þ responses Anti-VACV antibody, neutralization ND MV NAb

100%/mild

100%/mild

MV NAb

Anti-VACV antibody

VACV MV Nab, MPXV MV Nab, VACV comet inhibitory, MPXV comet inhibitory VACV: 100%/mild MV NAb, comet inhibitory ECTV: 100%/mild

100%/moderate

100%/mild

80%/ND

100%/mild

100%/ND

100%/significant

Anti-VACV antibody, Th1 response (IgG2a dominant)

91

51

53

80

52

79

79

79

78

92

53

100%/mild

Reference

*20%/severe

Weak MV NAb, comet inhibitory

Correlates of protection Survival/morbidity investigated

844 Cohen and Isaacs

DNA, Skin electroporation device, 3 doses DNA, gene gun (abdominal epidermis), 4 doses DNA, IM and ID, 3 doses

Protein, IM, 3 doses

DNA, IM and ID, 3 doses/ Boosted with protein, IM, 3 doses

Protein (expressed in baculovirus system), IM, 2 doses Replication incompetent rAd35 vector, 1 dose

VACV, NYBHCONN strain

VACV, WR strain

MPXV

MPXV

VACV, WR strain

VACV, WR strain

DNA, gene gun (abdominal epidermis), 4 doses DNA, gene gun (abdominal epidermis), 4 doses

VACV, WR strain

VACV, WR strain

MPXV

Delivery

Origin

None

None

BALB/c mice

BALB/c mice

BALB/c mice

BALB/c mice

CpG, alum

None

NHP

NHP

Protein in CpG

Alum or CpG

NHP

BALB/c mice

None

None

BALB/c mice

Animal model

none

Adjuvant

VACV, WR strain/IN/ 5E6 per mouse

VACV, WR strain/IP/ 5E7 per mouse

VACV, WR strain/IN/ *6E6 pfu/mo OR ECTV/IN/1000 pfu/mo VACV, WR strain/IP or IN/IP: 2E8 pfu/ mo, IN: 2E7 pfu/ mo

MPXV/IV/5E7 pfu

MPXV/IV/5E7 pfu

MPXV/IV/5E7 pfu

VACV, WR strain/IN/ 5E6/mouse

VACV, IHD-J/IN/ 2E6/muse

Challenge virus/ route/dose MV NAb, Th1/Th2 balanced response (IgG2a and IgG1 detected) MV NAb

66%/moderate

MV NAb

MV NAb, comet inhibitory, IP: 100%/mild Th1/Th2 balanced IN: 100%/mild to response (IgG2a and IgG1 moderate both produced), IFNg producers detected 100%/mild MV NAb

Weak Anti-VACV antibody, non-MV NAb, no CD8þ or CD4þ response 100%/mild to VACV MV NAb, MPXV MV severe NAb, MPXV EV NAb, CpG adjuvant gave a CD8þ and CD4þ response 100%/mild VACV MV NAb, MPXV MV NAb, MPXV EV NAb, Detectable CD8þ and CD4þ response (IL-2þ and IFNgþ/TNFaþ VACV: 100%/mild MV NAb, comet inhibitory ECTV: 100%/mild

0%/severe

26%/significant

100%/mild

Correlates of protection Survival/morbidity investigated

89

89

54

53

84

84

84

89

90

Reference

Abbreviations: alum, aluminum hydroxide; CPXV, cowpox virus (Brighton red strain); ECTV, ectromelia virus (Moscow strain); IHD-J, International Health Department strain J of VACV; ID, intradermal; IM, intramuscular; IP, intraperitoneal; IRM, immune response modifiers; IV, intravenous; MPXV, monkeypox (Zaire 79 strain); MV, mature virus; NAb, neutralizing antibody; ND, not done; NHP, nonhuman primate; NR, not reported; NYBH-CONN, New York Board of Health-Connaught strain; pfu/mo, plaque-forming unit per mouse; QS21, nontoxic saponin derived from the soapbark tree, Quillaja saponaria; rAd, recombinant adenovirus; Ribi, MPLþTDM (monophosphoryl lipid A þ trehalose dicorynomycolate) adjuvant; rVSV, recombinant Vesicular stomatitis virus; SC, subcutaneous; IN, intranasal; VACV, vaccinia virus; VARV, variola virus (India 1967 strain); VRP, Venezuelan equine encephalitis (VEE) virus replicon particle; WR, western reserve strain of vaccinia virus; IFNg, interferon gamma.

A27L, A33R, B5R, L1R, D8L

Genes

Table 2 (Continued )

Chapter 76: Improved Smallpox Vaccines 845

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protection from a lethal intranasal challenge with vaccinia virus in BALB/c mice with only mild disease (as measured by weight loss). This protection was achieved with as little as two doses given only two weeks apart and challenge of the mice three weeks after the boost vaccination (53). Addition of the A27 protein to the other three proteins to form a tetravalent vaccine provided little additional benefit in mice (53). This is a remarkable achievement considering that an immune response to only three proteins can provide protection from a virus that encodes 200 proteins. In addition to these four proteins, other MV targets of neutralizing antibodies such as D8 and H3 have been explored, though they have not been shown to greatly enhance the ability of the trivalent protein vaccine (A33, B5, L1) to protect against disease symptoms (77,78). The focus of protein subunit vaccination has mainly been on the envelope proteins of poxviruses that would target the infectious forms of the virus. However, poxviruses also encode a large assortment of nonstructural proteins that encode immune response modifiers (IRMs) (82). These proteins allow poxviruses to dampen or alter the immune response of the host to more efficiently spread throughout the host and ultimately infect the next host. Xu et al. (83) identified that the interferon (IFN) a/b binding protein encoded by the orthopoxvirus ectromelia virus (ECTV) EVM166 gene was critical for the efficient replication and spread of ECTV within its natural host, the mouse. With this in mind, they vaccinated mice with purified EVM166 protein to induce an antibody response that could neutralize the protein’s biological activity. They found that vaccinated mice challenged with a lethal dose of ECTV (by a footpad infection) were protected against death with only mildto-moderate disease symptoms (83). This was the first demonstration that a nonstructural protein could be used in a subunit vaccine to interfere with the ability of a virus to modulate the host immune response. This approach may be useful in future subunit smallpox vaccines, although it would be critical to determine which IRMs are most important for the replication and spread of smallpox. While subunit vaccines have shown protection from vaccinia virus challenge, it is also important to show the ability of a vaccine to protect against a viral challenge in its natural host. Thus, the ECTV (mousepox) challenge of mice has been a useful model since ECTV is a natural pathogen of the laboratory mouse (Mus musculus). Fang, et al. found that immunization with two doses of a single EV protein, A33, could partially protect BALB/c mice from death with a lethal dose of ECTV by footpad (47). By combining EV and MV targets, protein vaccinations with A33, B5, and L1 were able to fully protect against an intranasal ECTV challenge with only mild disease symptoms observed (53). A monkeypox model of poxvirus infection has also been studied using protein vaccination. This model is important because monkeypox represents a known human pathogen, and it is believed that if monkeys can be protected from monkeypox, it is likely that a similar immune response in humans could provide similar protection. Because of the expense of nonhuman primate studies and the need to have a model with a reproducible outcome of death in unvaccinated controls, the monkeypox model in nonhuman primates has focused on a high-dose intravenous challenge (34). There are obvious disadvantages of this model. One disadvantage is that the high-dose intravenous challenge bypasses the natural acquisition and spread of the virus in the host and is thought to reproduce mainly the stage of secondary viremia. Thus, this

type of challenge sets a very high hurdle for a vaccine to show protection, since natural acquisition of infection is likely caused by a much lower dose, which may be more easily controlled by vaccination. It is of equal concern that an intravenous challenge may accentuate the protection of vaccines that rely mainly on antibody responses that neutralize the incoming virus. Nevertheless, protein vaccination has been shown to protect monkeys from challenge. Heraud, et al., injected the monkeypox orthologs of A27, A33, B5, and L1 into rhesus macaques and found that these monkeys were completely protected from death with a lethal intravenous challenge with monkeypox, though they exhibited varying degrees of morbidity (84). Similarly, a small pilot study with the vaccinia virus A33, B5, and L1 proteins showed protection from severe disease after monkeypox challenge (80). Future studies using the monkeypox model will need to examine vaccine protection using more natural modes of challenge and will have to determine if adjustments in the vaccine formulation could enhance protection. Protein vaccination, in general, requires proper formulation to induce an effective immune response to the injected antigens. Varying the source of protein, amounts of protein, site of injection, and adjuvant can all play a role in the ability of the protein vaccination to elicit a potent and effective immune response. Live vaccinia virus vaccination with a fully protective vaccine such as Dryvax resulted in Th1-type cellular and humoral responses (52,85). For protein vaccination, appropriate adjuvants that skew the immune response toward a Th1-type response were shown to produce the best protection from both morbidity and death (53,80,81,84). While live vaccinia virus vaccination provides crossprotection against various orthopoxvirus infections, there is concern that a subunit smallpox vaccine based on vaccinia virus proteins might miss important epitopes present in the VARV ortholog proteins. Compared to live virus vaccination, the small differences of just a few amino acids between the vaccinia virus and VARV proteins may be amplified in a subunit vaccine that relies on just a few proteins to confer protection. For example, anti-B5 monoclonal antibodies have revealed that there are protective epitopes on the vaccinia B5 protein that are not present on the variola B5 ortholog (86). Similar findings have been reported with differences between the vaccinia virus A33 protein and the monkeypox A33 ortholog (71). Thus, another strategy that is being pursued by many groups is to use the VARV protein orthologs. For example, vaccination with smallpox orthologs of the vaccinia virus A27, B5, and D8 proteins provided complete protection from vaccinia virus challenge (79). Importantly, in this study it was found that the antibodies induced were at least as efficient at binding VARV protein as their vaccinia virus counterparts. Further studies will be needed to determine if VARV proteins can provide greater protection against smallpox virus than vaccinia virus proteins can confer.

DNA-Based Subunit Vaccines DNA vaccination involves the introduction of recombinant DNA plasmids that encode relevant protein antigens (87). The DNA plasmid is introduced into mammalian cells at the injection site, where the protein is then expressed. This is thought to have a number of advantages over simply vaccinating with a purified protein. (i) Using the normal host cell machinery to produce the protein, rather than using bacterial or baculovirus produced proteins, may create a more

Chapter 76: Improved Smallpox Vaccines

antigenically authentic protein to what would be made during a natural infection; (ii) By producing the protein within cells, it may generate a stronger T-cell response through normal major histocompatibility complex (MHC) class I presentation; (iii) Multiple gene targets can be easily included in a vaccine; and (iv) Lyophilized DNA can be stored at room temperature for long periods of time without degradation. These advantages have led a number of laboratories to pursue subunit DNA smallpox vaccines. Galmiche et al. were the first to demonstrate that protective responses could be generated by A33R or B5R DNA vaccines (65). Similar to what they found with protein vaccination, intramuscularly injecting DNA encoding either the A33 or B5 protein (but without the need for additional adjuvants) resulted in 100% survival of vaccinia virus challenged mice. This work led Hooper et al. to create a bivalent DNA vaccine encoding both the MV and EV proteins L1 and A33. Using a ‘‘gene gun,’’ a device created to inject DNA coated on gold beads, they were able to show 100% survival and only mild disease symptoms after challenge by the intraperitoneal route with a lethal dosage of vaccinia virus (50). Hooper et al. later expanded upon this work by including the A27L and B5R genes to make a tetravalent vaccine (51). While they found that a bivalent A27L and B5R DNA vaccine did not give complete protection, the tetravalent vaccine (A27L, A33R, B5R, and L1R) gave complete protection from a lethal intraperitoneal vaccinia virus challenge with only mild disease symptoms (51). Pulford et al. used DNA vaccines against single MV and EV vaccinia virus protein targets to determine if they could provide protection from an intranasal challenge with vaccinia virus (88). In addition to showing that the B5R DNA vaccine offered 100% protection from challenge, they also demonstrated that smallpox subunit DNA vaccines could induce an IFNg response and a memory response mediated by a CD4þ T-cell population (88). To determine if additional antigen targets would be beneficial in a polyvalent vaccine, Sakhatskyy et al. (89) added a fifth gene, D8L, to the tetravalent DNA vaccine formulation used by Hooper, et al. (51). They found that adding the D8L gene to the other four vaccinia virus genes offered better protection in an intranasal model of challenge than without it, though protection was not 100% (89). Additionally, Sakhatskyy et al. determined that using the VARV homologs of A27L, B5R, and D8L partially protected mice from a lethal intranasal vaccinia virus challenge (79). As discussed previously, the additional use of VARV sequence to construct a vaccine may be important to ensure immune reactivity in the face of a smallpox challenge. The lack of complete protection from morbidity seen by some investigators with the polyvalent DNA vaccines (Table 2) could be due to the method of DNA delivery and the type of immune response that was generated. To determine if this was the case, Hooper et al. used a novel method of skin electroporation to deliver their DNA vaccine (90). They found that this method of delivery improved the efficacy of their tetravalent DNA vaccine (A27L, A33R, B5R, and L1R), and provided complete protection from challenge in an intranasal model of infection. They found that skin electroporation mimicked to a greater extent the type of antibodies produced during Dryvax vaccination, by inducing more mouse IgG2a antibodies (Th1 response), than the gene gun method of DNA delivery (90). This finding makes the skin electroporation method a more attractive method of DNA delivery than the gene gun method. The mode of vaccination was further highlighted by work in nonhuman primates. Hooper et al. found that they could

847

generate complete protection from an intravenous monkeypox virus challenge using their tetravalent (A27L, A33R, B5R, and L1R) formulation delivered by a gene gun (91). However, Heraud et al. found that when the monkeypox homologs of A27L, A33R, B5R, and L1R were injected as naked DNA, there was no protection from monkeypox challenge (84). Going forward, smallpox DNA vaccines will need to be administered in a way best able to generate a Th1-type immune response that includes both neutralizing antibodies and strong T-cell responses.

Vector-Based Subunit Vaccines Vectored vaccines utilize a nonpathogenic virus or bacteria to deliver a desired antigen. Because protein and DNA vaccinations have been shown to require multiple vaccinations to achieve protective immunity, vectored vaccines have been pursued as a way to generate a smallpox vaccine that can offer protection in a single vaccination. In a smallpox outbreak setting, it would be important to induce protective immunity as rapidly as possible to avoid spread of the virus. The first laboratory to explore vector subunit smallpox vaccines utilized replicon particles of Venezuelan equine encephalitis virus (92). By expressing A27, A33, and B5, they generated a strong mouse IgG2a antibody response (Th1-type response), and protected mice from a sublethal dose of cowpox virus. Kaufman et al. utilized replication incompetent recombinant Adenovirus serotype 35 (rAd35) vectors expressing A27, A33, B5, and L1 antigens (54). By delivering a single immunization with all four rAd35 vectors, they were able to achieve complete protection in mice from a lethal intranasal vaccinia virus challenge. The rAd35 vaccine generated strong MV neutralizing antibodies that were balanced between mouse IgG2a and IgG1 antibodies (Th1 and Th2 response) (54). Vectored vaccines so far appear to be a promising delivery method for subunit smallpox vaccines, but much work is still needed to determine the immunogenicity and safety profile in nonhuman primates and humans.

CONCLUSIONS While stockpiling of a live vaccinia virus vaccine grown in cell culture has been successful, significant concerns about the minor and major complications from this vaccine remain, especially in populations that have contraindications for vaccination. More attenuated live vaccinia virus vaccines, which will be much safer to give to a diverse population, will likely be the next new generation smallpox vaccine that gains regulatory approval. However, growing and maintaining a stock of a live virus vaccine, as well as the potential for adverse events, are limitations that fuel the continued pursuit of future generation smallpox vaccines. Subunit vaccines are showing great success. Many possibilities for protective vaccines exist, and future efforts to directly compare different vaccination strategies will be needed. For example, Barefoot et al. chose a single immunogen, B5, and compared multiple vaccination strategies for generating immune responses and examined the level of protection from challenge (93). They found that a heterologous prime-boost combination of recombinant vesicular stomatitis virus (rVSV) expressing B5 and recombinant Venezuelan equine encephalitis virus replicons (VRP) expressing B5 as the most synergistic regimen. A possible scenario is that the best protection from challenge may incorporate a combination

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of strategies, such as a DNA prime and protein boost. Heraud et al. found that this particular strategy offered the best protection from a monkeypox challenge when compared to either DNA or protein vaccination alone (84). While subunit vaccines have mainly focused on production of antibody responses, subunit smallpox vaccines expressing vaccinia virus immunodominant T-cell epitopes have been shown to protect mice from orthopoxvirus challenge (94). Thus, another strategy is to identify epitopes that are critical for T cell– mediated protection from smallpox and to provide these to the host immune system (95). Many of the current subunit vaccines under development have only been shown to produce short-term protection from challenge in mice, on the order of three to four weeks after the last vaccine dose. Two studies have examined the ability of a subunit smallpox vaccine to protect long-term (3–6 months after the final vaccine dose) against lethal (and sublethal) challenge with vaccinia virus. These studies showed that protection against death was incomplete and mice developed significant to severe symptoms (53,93). Live vaccinia virus vaccination offers long-term immunity, on the order of decades (96) that likely may protect against death from smallpox (97). Subunit vaccines will likely not achieve this impressive benchmark, but may be improved with continued formulation refinement. To protect against smallpox outbreaks that may be many months or years apart, it may be necessary to boost individuals who were previously vaccinated with only a subunit vaccine. An alternative strategy might be to use subunit vaccines as a way to prime diverse populations to allow safer vaccination with live vaccinia virus vaccines (98).

REFERENCES 1. Fenner F, Henderson DA, Arita I, et al. Smallpox and Its Eradication. 1st ed. Geneva: World Health Organization, 1988. 2. Breman JG, Henderson DA. Poxvirus dilemmas—monkeypox, smallpox. N Engl J Med 1998; 339:556–559. 3. Atlas RM. The threat of bioterrorism returns the fear of smallpox. Curr Opin Microbiol 1998; 1:719–721. 4. Hutin YJ, Williams RJ, Malfait P, et al. Outbreak of human monkeypox, democratic republic of congo, 1996 to 1997. Emerg Infect Dis 2001; 7(3):434–438. 5. Nalca A, Rimoin AW, Bavari S, et al. Reemergence of monkeypox: prevalence, diagnostics, and countermeasures. Clin Infect Dis 2005; 41(12):1765–1771. 6. Reed KD, Melski JW, Graham MB, et al. The detection of monkeypox in humans in the Western Hemisphere. N Engl J Med 2004; 350(4):342–350. 7. Rosenthal SR, Merchlinsky M, Kleppinger C, et al. Developing new smallpox vaccines. Emerg Infect Dis 2001; 7(6):920–926. 8. Poland GA. Smallpox vaccines: from first to second to third generation. Lancet 2005; 365(9457):362–363. 9. Wiser I, Balicer RD, Cohen D. An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies. Vaccine 2007; 25(6):976–984. 10. Artenstein AW. New generation smallpox vaccines: a review of preclinical and clinical data. Rev Med Virol 2008; 18(4):217–231. 11. Kretzschmar M, Wallinga J, Teunis P, et al. Frequency of adverse events after vaccination with different vaccinia strains. PLoS Med 2006; 3(8):e272. 12. Weltzin R, Liu J, Pugachev KV, et al. Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 2003; 9(9): 1125–1130; [Epub August 17, 2003]. 13. Monath TP, Caldwell JR, Mundt W, et al. ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)—a second-generation smallpox vaccine for biological defense. Int J Infect Dis 2004; 8(suppl 2):31–44.

14. Marriott KA, Parkinson CV, Morefield SI, et al. Clonal vaccinia virus grown in cell culture fully protects monkeys from lethal monkeypox challenge. Vaccine 2008; 26(4):581–588. 15. Greenberg RN, Kennedy JS. ACAM2000: a newly licensed cell culture-based live vaccinia smallpox vaccine. Expert Opin Investig Drugs 2008; 17(4):555–564. 16. CDC. Notice to Readers: Newly licensed smallpox vaccine to replace old smallpox vaccine. MMWR Morb Mortal Wkly Rep 2008; 57(8):207–208. 17. Weiner DB. Keeping the lock on smallpox. Nat Med 2003; 9(9): 1115–1116. 18. Poland GA, Grabenstein JD, Neff JM. The US smallpox vaccination program: a review of a large modern era smallpox vaccination implementation program. Vaccine 2005; 23(17–18):2078–2081. 19. CDC. Recommendations for using smallpox vaccine in a pre-event vaccination program. MMWR Morb Mortal Wkly Rep 2003; 52(RR07): 1–16. 20. Hashizume S. A new attenuated strain Lc16m8 of vaccini virus for safer smallpox vaccination. Clin Virol 1975; 3:229–235. 21. Hashizume S, Yoshizawa H, Morita M, et al. Properties of attenuated mutant of vaccinia virus, LC16m8 derived from Lister strain. In: Quinnen J, ed. Vaccinia Viurses as Vecors for Vaccine Antigens. New York: Elsevier, 1985:87–100. 22. Kenner J, Cameron F, Empig C, et al. LC16m8: an attenuated smallpox vaccine. Vaccine 2006; 24(47–48):7009–7022. 23. Takahashi-Nishimaki F, Funahashi S, Miki K, et al. Regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins. Virology 1991; 181:158–164. 24. Empig C, Kenner JR, Perret-Gentil M, et al. Highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge. Vaccine 2006; 24(17):3686–3694. 25. Saijo M, Ami Y, Suzaki Y, et al. LC16m8, a highly attenuated vaccinia virus vaccine lacking expression of the membrane protein B5R, protects monkeys from monkeypox. J Virol 2006; 80(11):5179–5188. 26. Kidokoro M, Tashiro M, Shida H. Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8. Proc Natl Acad Sci U S A 2005; 102(11): 4152–4157. 27. Bell E, Shamim M, Whitbeck JC, et al. Antibodies against the extracellular enveloped virus B5R protein are mainly responsible for the EEV neutralizing capacity of vaccinia immune globulin. Virology 2004; 325(2):425–431. 28. Putz MM, Midgley CM, Law M, et al. Quantification of antibody responses against multiple antigens of the two infectious forms of Vaccinia virus provides a benchmark for smallpox vaccination. Nat Med 2006; 12(11):1310–1315. 29. Mayr A. Smallpox vaccination and bioterrorism with pox viruses. Comp Immunol Microbiol Infect Dis 2003; 26(5–6):423–430. 30. Slifka MK. The future of smallpox vaccination: is MVA the key? Med Immunol 2005; 4(1):2. 31. Davies DH, Wyatt LS, Newman FK, et al. Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus Ankara is comparable to that of Dryvax. J Virol 2008; 82(2):652–663. 32. Wyatt LS, Earl PL, Eller LA, et al. Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge. Proc Natl Acad Sci U S A 2004; 101(13):4590–4595. 33. Phelps AL, Gates AJ, Hillier M, et al. Comparative efficacy of modified vaccinia Ankara (MVA) as a potential replacement smallpox vaccine. Vaccine 2007; 25(1):34–42. 34. Earl PL, Americo JL, Wyatt LS, et al. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against monkeypox. Nature 2004; 428:182–185. 35. Stittelaar KJ, van Amerongen G, Kondova I, et al. Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus. J Virol 2005; 79(12):7845–7851. 36. Earl PL, Americo JL, Wyatt LS, et al. Recombinant modified vaccinia virus Ankara provides durable protection against disease

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59. Schmelz M, Sodeik B, Ericsson M, et al. Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network. J Virol 1994; 68:130–147. 60. Rodriguez JF, Janeczko R, Esteban M. Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus. J Virol 1985; 56:482–488. 61. Czerny CP, Mahnel H. Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies. J Gen Virol 1990; 71(pt 10):2341–2352. 62. Gordon J, Mohandas A, Wilton S, et al. A prominent antigenic surface polypeptide involved in the biogenesis and function of the vaccinia virus envelope. Virology 1991; 181:671–686. 63. Wolffe EJ, Vijaya S, Moss B. A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies. Virology 1995; 211:53–63. 64. Ichihashi Y, Oie M. Neutralizing epitope on penetration protein of vaccinia virus. Virology 1996; 220:491–494. 65. Galmiche MC, Goenaga J, Wittek R, et al. Neutralizing and protective antibodies directed against vaccinia virus envelope antigens. Virology 1999; 254:71–80. 66. Hsiao JC, Chung CS, Chang W. Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells. J Virol 1999; 73(10):8750–8761. 67. Lin CL, Chung CS, Heine HG, et al. Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo. J Virol 2000; 74(7):3353–3365. 68. Aldaz-Carroll L, Whitbeck JC, Ponce de Leon M, et al. Epitopemapping studies define two major neutralization sites on the vaccinia virus extracellular enveloped virus glycoprotein B5R. J Virol 2005; 79(10):6260–6271. 69. Aldaz-Carroll L, Whitbeck JC, Ponce de Leon M, et al. Physical and immunological characterization of a recombinant secreted form of the membrane protein encoded by the vaccinia virus L1R gene. Virology 2005; 341(1):59–71. 70. Lustig S, Fogg C, Whitbeck JC, et al. Combinations of polyclonal or monoclonal antibodies to proteins of the outer membranes of the two infectious forms of vaccinia virus protect mice against a lethal respiratory challenge. J Virol 2005; 79(21):13454–13462. 71. Golden JW, Hooper JW. Heterogeneity in the A33 protein impacts the cross-protective efficacy of a candidate smallpox DNA vaccine. Virology 2008; 377(1):19–29. 72. Davies DH, Liang X, Hernandez JE, et al. Profiling the humoral immune response to infection by using proteome microarrays: high-throughput vaccine and diagnostic antigen discovery. Proc Natl Acad Sci U S A 2005; 102(3):547–552. 73. Jones-Trower A, Garcia A, Meseda CA, et al. Identification and preliminary characterization of vaccinia virus (Dryvax) antigens recognized by vaccinia immune globulin. Virology 2005; 343(1): 128–140. 74. He Y, Manischewitz J, Meseda CA, et al. Antibodies to the A27 protein of vaccinia virus neutralize and protect against infection but represent a minor component of Dryvax vaccine-induced immunity. J Infect Dis 2007; 196(7):1026–1032. 75. Lai CF, Gong SC, Esteban M. The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia-coli induces virus immunity in animals. J Virol 1991; 65(10):5631–5635. 76. Lustig S, Fogg C, Whitbeck JC, et al. Synergistic neutralizing activities of antibodies to outer membrane proteins of the two infectious forms of vaccinia virus in the presence of complement. Virology 2004; 328(1):30–35. 77. Davies DH, McCausland MM, Valdez C, et al. Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice. J Virol 2005; 79(18):11724–11733. 78. Berhanu A, Wilson RL, Kirkwood-Watts DL, et al. Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus

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89. Sakhatskyy P, Wang S, Chou TH, et al. Immunogenicity and protection efficacy of monovalent and polyvalent poxvirus vaccines that include the D8 antigen. Virology 2006; 355(2): 164–174. 90. Hooper JW, Golden JW, Ferro AM, et al. Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge. Vaccine 2007; 25(10): 1814–1823. 91. Hooper JW, Thompson E, Wilhelmsen C, et al. Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J Virol 2004; 78(9):4433–4443. 92. Thornburg NJ, Ray CA, Collier ML, et al. Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge. Virology 2007; 362(2):441–452. 93. Barefoot B, Thornburg NJ, Barouch DH, et al. Comparison of multiple vaccine vectors in a single heterologous prime-boost trial. Vaccine 2008; 20:20. 94. Snyder JT, Belyakov IM, Dzutsev A, et al. Protection against lethal vaccinia virus challenge in HLA-A2 transgenic mice by immunization with a single CD8þ T-cell peptide epitope of vaccinia and variola viruses. J Virol 2004; 78(13):7052–7060. 95. Tscharke DC, Karupiah G, Zhou J, et al. Identification of poxvirus CD8þ T cell determinants to enable rational design and characterization of smallpox vaccines. J Exp Med 2005; 201(1):95–104. 96. Hammarlund E, Lewis MW, Hansen SG, et al. Duration of antiviral immunity after smallpox vaccination. Nat Med 2003; 9(9): 1131–1137. 97. Hanna W, Baxby D. Studies in smallpox and vaccination. 1913. Rev Med Virol 2002; 12(4):201–209. 98. Otero M, Calarota SA, Dai A, et al. Efficacy of novel plasmid DNA encoding vaccinia antigens in improving current smallpox vaccination strategy. Vaccine 2006; 24(21):4461–4470.

77 Anthrax Vaccines Arthur M. Friedlander U.S. Army Medical Research Institute of Infectious Diseases, Frederick; and Uniformed Services University of the Health Sciences School of Medicine, Bethesda, Maryland, U.S.A.

Stephen F. Little U.S. Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, U.S.A.

Marc Gurwith PaxVax, San Diego, California, U.S.A.

INTRODUCTION Natural infection of humans by Bacillus anthracis, the etiological agent of anthrax, generally results from contact with infected animals or spore-contaminated animal products. Because of the rarity of human anthrax, the only interest for developing anthrax vaccines for humans is to protect against the use of B. anthracis as a bioweapon to intentionally cause disease. The outbreak of inhalational anthrax in the former Soviet Union in 1979 from the accidental release of spores (1), the admission that Iraq had produced anthrax spores as weapons (2), and the recent successful use of B. anthracis to cause disease in the autumn of 2001 (3) have highlighted the need for improved anthrax vaccines. The manifestations of the disease vary depending upon the route of exposure. Spores that enter through abrasions in the skin cause cutaneous anthrax while ingestion results in gastrointestinal anthrax and aerosol exposure to spores causes inhalational anthrax. Spores are thought to germinate either within phagocytes or extracellularly, perhaps depending upon the route of infection. The major virulence determinants of B. anthracis are the poly-g-D-glutamic acid (gDPGA) capsule and the binary exotoxins, lethal toxin, and edema toxin. Several additional factors were recently described that are necessary for full virulence including a siderophore (4) and a manganese ABC (ATP-binding cassette) transporter (5). The gDPGA capsule, encoded on a 96-kb plasmid (pXO2) (6,7), enhances virulence by protecting the bacterium from phagocytosis and lysis by cationic serum proteins (8). The establishment of disease is further enhanced by lethal toxin and edema toxin, which interfere with the function of cells of the innate immune system (9–12) allowing the infection to progress. Unlike other binary toxins, anthrax toxins possess a separate cell-binding domain [protective antigen (PA)], which binds to cell receptors, and two separate enzymatic molecules, lethal factor (LF) and edema factor (EF). All three components are encoded on a second 182-kb plasmid (pXO1) (13). PA, originally identified as a protein conferring protection against experimental anthrax in animals (14), was only later shown to be the critical cell receptor–binding component of the exotoxins. The crystal structure of PA identified four domains associated with different functions of the toxin, such as cell receptor binding,

oligomerization, membrane insertion, and translocation (15). Antibodies to PA have been shown to be protective and anti-LF and anti-EF antibodies can also be important in protection (16) as LF and EF each possess a domain that interacts with PA and a domain for biochemical activity. PA is activated by furin or other proteases on the cell surface by cleavage at a trypsinsensitive site after binding to cell-surface receptors or perhaps by proteases present in plasma, releasing a 20-kDa N-terminal fragment. Heptameric oligomers are formed by the 63-kDa fragment (PA63) bound to the receptor that competitively binds LF and/or EF to form lethal or edema toxin, respectively. Lethal and edema toxins are endocytosed by the cells and LF and EF are delivered to the cytosol where they exert their physiological effects (17). Although EF is an adenylate cyclase causing pharmacological elevation of cyclic adenosine monophosphate (cAMP) resulting in cell dysfunction (18), and LF is a metalloprotease that targets mitogen-activated protein kinase kinase (MAPKK) leading to disruption of cell signal transduction (19,20), the site of action and the role of the toxins and other virulence factors and mechanisms of death from infection remain obscure. The pathophysiology of anthrax is due to an overwhelming bacteremia, widespread infection of multiple organs, and the actions of the toxins that result in respiratory failure with pulmonary edema and pleural effusions, often associated with meningitis and subarachnoid hemorrhage.

IMMUNITY Natural infection-derived immunity to anthrax is thought to occur based on anecdotal evidence that reinfections in humans are rare and less severe (21); however, the data are very limited. Perhaps the best evidence comes from recent studies showing that nonhuman primates that have recovered after treatment for inhalational anthrax are resistant to reinfection (22), and from older data in other animals (23). Both PA-based protein and live attenuated spore vaccines are effective in experimental animals (see below). The mechanism of immunity is thought to be humoral, as passive protection against both parenteral and aerosol challenge with immune serum has been clearly demonstrated with both PA-based and live attenuated spore vaccines

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(24–26). There is compelling evidence that protection induced by PA vaccines in the guinea pig is due to humoral immunity based on similar levels of circulating antibody being required for both active and passive protection against intradermal (ID) challenge (27), and similar findings were reported for rabbits given an intranasal (IN) challenge (28). Furthermore, PA vaccine-induced protection in mice against an unencapsulated toxinogenic strain could be transferred with immune serum but not with spleen cells (29). This research has led to the search for serological correlates of immunity to PA-based vaccines using antibodies to PA. In general, antibodies to PA correlate with immunity in mice (16), guinea pigs (27), rabbits (28,30,31), and nonhuman primates (32). In addition to using an ELISA to measure antibody levels, a toxin-neutralizing assay (TNA) is available to evaluate the functional ability of PA and LF antibodies to interfere with lethal toxin formation (33). Antibodies directed against domains of PA or LF involved in toxin formation will neutralize lethal toxin cytotoxicity. Under some circumstances, measurement of the toxin-neutralizing activity of anti-PA antibodies that block binding of PA to the host cell receptor and LF binding to PA have correlated better with protection than antibodies determined by ELISA (27).

Given the protection afforded experimental animals by passive transfer of antibodies to either PA-based or live spore vaccines, there has been assumed to be little role for cellmediated immunity in protection by these vaccines. The only evidence for cell-mediated immunity was the demonstration that the enhanced protection against a nontoxinogenic encapsulated strain in mice afforded by adding formaldehyde-inactivated spores to a PA vaccine (34) appears to be mediated by CD4 T lymphocytes and not humoral immunity (35). Nonhuman primates that develop anti-PA antibodies after either PA vaccination (36) or infection (22) are immune. However, immunity resulting from infection may not be entirely based upon the presence of antibodies to PA. Protection against infection can be enhanced by including live (37) or killed spores with PA as noted above, and in some animals with killed spores alone (34), suggesting that spore and/or other bacillus antigens may contribute to protection. Currently, prevention is provided by two anthrax vaccines licensed for use in humans that consist of aluminum adjuvanted culture filtrates from attenuated strains. Table 1 contains a summary of the different strategies to develop new anthrax vaccines, to enhance immune responses with

Table 1 Summary of New Vaccines and Strategies Against Anthrax Vaccine type

Composition of vaccine

Vaccination route

Rationale for usage

Referencea

Cell-free vaccines

AVA (V770-NP1-R, toxinogenic, unencapsulated strain filtered culture supernatant) AVA with CpG oligodeoxynucleotide rPA from various sources (B. anthracis, E. coli, B. subtilis, baculovirus, plants) adsorbed to aluminum adjuvant rPA combined with various adjuvants rPA combined with various adjuvants or carriers rPA combined with various adjuvants or carriers rPA combined with LF and/or EF

IN

Stimulate mucosal immunity with licensed vaccine

63

SC, IM

Enhance immunity

60,61

SC, IM

Known composition; two currently in clinical trials

36,80,155,161,162

IM, SC

Enhance immunity

80,81,83

IN

Alternative delivery system and stimulation of mucosal immunity Alternative delivery system

84,107,108, 112–114,117,119 16,84,120,121

Enhance immunity to toxin by including other components Potential to reduce toxicity when given with LF or EF or during infection Multi-component vaccine to enhance immunity by targeting toxin and capsule (bacillus) Induce immunity to toxins and other bacterial proteins; may stimulate CMI Reduce toxicity of vaccine; enhanced expression of PA

45,89,90

rPA-based vaccines

Live spore vaccines

Live bacterial vaccines Genetic

Transcutaneous IM

rPA mutants

IP, IM

gDPGA capsule with rPA or gDPGA conjugate

SC

Toxinogenic, unencapsulated strains

Parenteral

Attenuated unencapsulated recombinant strains containing native or mutated PA or PA plus mutated LF/EF Attenuated recombinant strains

Parenteral

Oral

Salmonella strains expressing PA

Oral

Recombinant viral vectors

IM

plasmid DNA

IM, Electroporation

a References are representative and not all inclusive. Abbreviations: IN, intranasal; SC, subcutaneous; IM, intramuscular; IP, intraperitoneal.

Alternative delivery system and stimulation of mucosal immunity Alternative delivery system and stimulation of mucosal immunity Enhance immunity; may stimulate CMI Ease of production; may stimulate CMI

93–95

98–100

90,123,134

128–130

132 79,138,139 87,140–142 91,145,149

Chapter 77: Anthrax Vaccines

adjuvants, and to explore alternative ways (e.g., intranasal, transcutaneous) to deliver anthrax vaccine antigens.

CELL-FREE CULTURE SUPERNATANT VACCINES Early anthrax studies identified an immunizing fraction, termed PA in the United States and factor II in the United Kingdom, in extracts from edematous anthrax lesions of guinea pigs (14,38,39) and in culture supernatants of B. anthracis (40–43). Two additional components were also identified, LF or factor I, and EF or factor III, which formed lethal toxin and edema toxin, respectively, when combined with PA (44,45), as described above. The first cell-free anthrax vaccine tested for safety and efficacy in humans employed in woolen mills (46,47) was developed by Wright et al. (41,46) who precipitated filtered aerobic culture supernatants of a nonproteolytic mutant of B. anthracis, R1-NP, with alum. Improvements to the alum vaccine were made by adapting anaerobic cultural growth conditions, to allow for an increase in the scale of manufacture, and by selecting a more productive B. anthracis strain (48,49). The anthrax vaccine in the United States, anthrax vaccine adsorbed (AVA; also referred to as AVA BioThrax, MDPH, and MDPH-PA in the literature), licensed in 1970, is prepared by adsorbing filtered microaerophilic culture supernatant fluids from the toxinogenic unencapsulated B. anthracis V770-NP1-R strain onto aluminum hydroxide gel (1.2 mg/mL of aluminum). The final product contains benzethonium chloride (25 mg/mL) as a preservative and formaldehyde (100 mg/mL) as a stabilizer. AVA is administered subcutaneously (SC) (0.5 mL) initially as three biweekly injections followed by three injections at 6, 12, and 18 months, and yearly boosters thereafter. The licensed anthrax vaccine used in the United Kingdom, anthrax vaccine precipitated (AVP), is prepared by precipitating aerobic culture filtrates of the unencapsulated toxinogenic Weybridge strain with alum and adding thiomerosal as a preservative (42,50). AVP is administered in four doses (0.5 mL each), three at intervals of three weeks and the fourth six months after the third dose, with yearly boosters thereafter. The antigens adsorbed by the adjuvant in AVP or AVA, characterized by either serological titer, immunoblot analysis, or mass spectrophotometry of proteins from two-dimensional gel electrophoresis (51–56), show the presence of PA and smaller amounts of LF and EF, as well as other proteins. AVP is reported to contain about 40 mg/mL of PA (57). The vaccination schedule and SC route of injection used for humans were based upon efficacy studies with guinea pigs and nonhuman primates (47,58). Several studies in animals have been performed in which the immune response to AVA or AVP was enhanced by adding various adjuvants (59–62) or administered intranasally (63). Humans show an increased serological response by the addition of adjuvants (see below).

RECOMBINANT PA–BASED VACCINES The current licensed vaccine, AVA, has met U.S. Food and Drug Administration (FDA) standards for safety and efficacy (64,65) and was considered in a review in 2002 by the National Academy of Sciences to be safe and effective for protection of humans against anthrax, including inhalational anthrax (65). Nevertheless, concerns regarding its undefined composition, variability in lot-to-lot antigen composition, extensive dosing regimen, and local reactogenicity have been the impetus for developing a well-defined vaccine based upon PA (Table 1).

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Recombinant PA has been purified mainly from B. anthracis (66,67), B. subtilis (68,69), and Escherichia coli (70), as well as from other expression systems, including baculovirus (71) and plants (72–74). One or two doses of PA given with aluminum hydroxide have been shown to protect nonhuman primates against an aerosol challenge with a fully virulent encapsulated strain (36,75,76). A few studies have been conducted examining for key PA regions. Vaccination with the cleaved cell-bound form of PA, PA63, at doses of 5 and 50 mg, was reported to protect rabbits against aerosol challenge with a fully virulent encapsulated strain at a lower but not statistically significant different rate than did AVA (77). The same study also showed that both PA63 and AVA gave some protection against aerosol challenge to nonhuman primates, although small numbers of animals were used (77). Mice vaccinated with the cell receptor– binding domain (domain 4) of PA were protected against infection with an unencapsulated toxinogenic strain, but it was not established if this was equivalent to the protection observed with full-length PA (78,79).

PA Combined with Adjuvants in Parenteral Vaccines In addition to the aluminum-based adjuvants, aluminum hydroxide gel (aluminum oxyhydroxide), aluminum phosphate gel (aluminum hydroxyphosphate), or potassium aluminum sulfate (alum), which are approved for licensed vaccines, numerous experimental adjuvants have been examined for their ability to increase the antibody titer to PA and to enhance protection against infection in mice and guinea pigs (59,80–82), animals that have proved difficult to protect against challenge with fully virulent encapsulated B. anthracis strains. A considerable increase in the TNA titer of mice was measured when PA was combined with either CpG oligodeoxynucleotides (ODN) alone or combined with Pluronic F127, a nonionic block copolymer compared with Pluronic F127 or alum alone, or Pluronic F127 and chitosan, an IgA inducer (83). Varying effects on the serological response to PA have been observed in different animal models inoculated by different vaccination routes when CpG is formulated with PA (84) or AVA (85). Adding CpG to AVA improved the antibody response to PA in mice (85), guinea pigs (61), and nonhuman primates (85) and increased protection for mice against challenge with an unencapsulated toxinogenic strain (85) and for guinea pigs against challenge with a fully virulent encapsulated strain after a single dose of vaccine (61). The only experimental adjuvant that has been used with PA clinically is CpG (see below). In another study using nonhuman primates, PA given with either aluminum hydroxide gel or the adjuvants saponin QS-21 or monophosphoryl lipid A (MPL) gave protection against a lethal aerosol challenge (36), although none of the adjuvant tested gave better protection than aluminum hydroxide. Another recent approach to enhance immunogenicity of PA was to modify the molecule to direct it to antigen-presenting cells possessing complement receptor 2. Vaccination with PA cross-linked to a monoclonal antibody to complement receptor 2 resulted in increased antibody titer and some increase in protection of mice against a lethal toxin challenge compared with vaccination with PA alone, although comparisons to PA given with aluminum adjuvants were not reported (86). Other attempts to increase the immunogenicity have involved the use of bacteriophage T4 particles displaying PA

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and the other toxin components. Fusions of the nonessential, highly antigenic outer capsid protein (87) or the nonessential, small outer capsid protein (88) with PA, LF, and EF either individually or in combination were assembled in vitro on the surface of bacteriophage T4 as a multicomponent anthrax vaccine. Mice inoculated with bacteriophage T4 displaying PA, LF, and EF elicited strong antibody responses against all three antigens and lethal toxin-neutralizing antibodies, although no protection studies have been reported. The phage particle stimulated an immune response in the absence of any added adjuvants.

Interaction of PA with LF and EF While PA is the major protective toxin component, there have been varying results as to whether LF or EF contributes to immunity of PA. Older studies showed some increase in PAinduced protection against infection with EF (45), while others showed an additional benefit with LF but not with EF (89), and some showed no added benefit of either LF or EF despite a good immune response (90). These different results may relate to the purity of the proteins and whether the toxins were biologically active. LF expressed on a plasmid has been reported to confer limited protection against aerosol challenge in rabbits exposed to virulent encapsulated anthrax with a delay in time to death but no significant increase in survival compared with the animals given a control plasmid (91) (see below). In addition, the N-terminal fragment of EF expressed on a replication-incompetent adenoviral vector offered partial protection to mice against challenge with an unencapsulated toxinogenic strain, suggesting some benefit, although a control vector without EF was not used (92). Antibodies induced to EF were cross reactive with LF and so also inhibited lethal toxin activity. Mutations of PA have been constructed to address concerns regarding the possible cytotoxicity resulting from the interaction of biologically active PA, LF, and EF if used together in a vaccine formulation, although this may be unlikely when they are mixed with an adjuvant. Both PA163–168, a mutant with deletion of the furin/trypsin cleavage site rendering it unable to interact with LF or EF, and PA313–314, a mutant with deletion at a chymotrypsin cleavage site, making it defective in translocation, protected guinea pigs from challenge with a virulent encapsulated strain, although quantitative comparisons with native PA were not reported (93,94). The lower toxin-neutralizing antibody titers produced by the furin/trypsin site mutant compared with native PA, although not significant, suggest that antibodies directed against the LF/EF binding site, in addition to those directed against the cell-receptor domain on PA may contribute to toxin neutralization (94). Mice injected with PAK397D D425K, a nontoxic dominant-negative toxin inhibitor that blocks translocation of toxin into the cytosol, had higher levels of anti-PA IgG than those given native PA. Both protected mice against a lethal toxin challenge, although these studies did not determine if the mutant gave greater protection than native PA (95). It has been suggested that such a nontoxic PA mutant might be used postexposure both as an antitoxin and a vaccine. Mice vaccinated with spores of mutants of the unencapsulated toxinogenic B. anthracis Sterne strain deficient in the expression of one or two toxin components also demonstrated some variable degree of protection against challenge with an unencapsulated toxinogenic strain in the absence of PA (96),

suggesting that LF and EF or factors on pX01 may contribute to protection. B. anthracis Sterne strains expressing a mutation in LF (PA, EF, LFH686A) or a deletion of EF (PA, DEF, LFH686A) also protected guinea pigs against challenge with a virulent encapsulated strain (97), although quantitative comparisons with Sterne were not reported. These mutant strains are expected to be less reactogenic than Sterne. All these reports suggest a potential role for mutant PA, LF, or EF as immunogens in a PAbased vaccine.

OTHER ANTIGENS

The gDPGA capsule is a T-independent antigen inducing predominantly IgM antibodies, and is converted to a T-dependent antigen giving an IgG response after coupling to a carrier protein (98). The first demonstration that gDPGA could contribute to protection against a lethal challenge was reported by Chabot et al. (99) in which gDPGA combined with PA and the adjuvants MPL and trehalose dimycolate (TDM) gave significantly greater protection than either gDPGA or PA alone against an SC challenge with a virulent encapsulated strain in mice, while no protection occurred using gDPGA cross-linked to a protein carrier. Improvements in cross-linking procedures were reported by Joyce et al. (100) who covalently coupled gDPGA to the outer membrane protein complex of Neisseria meningitidis serotype B and observed protection in the mouse model without added PA. Other workers noted increased IgG antibodies to gDPGA by cross-linking capsule or glutamate peptides to protein carriers, but no protection studies have been reported (101–103). Conjugating gDPGA to a PA-dominant negative inhibitor elicited higher anti-PA and anti-gDPGA titers in mice than did native PA alone, but no studies of protection against infection were reported (95). Monoclonal antibodies to gDPGA have also been shown to be protective (101). These results suggest that adding capsule antigens to a PA vaccine may offer additional benefit. The long-reported enhanced protection afforded by live unencapsulated toxinogenic spore vaccines compared with PAbased protein vaccines coupled with their much lower antitoxin antibody response has suggested that antigens other than PA and capsule may contribute to protection. This is supported by experimental evidence that formaldehyde-inactivated spores enhance the protection afforded by PA in some animal models (34). The nature of the spore antigens contributing to protection is unknown, but it does not appear to be related to the major exosporium protein, BclA (M. Mock, personal communication). Other workers have reported a modest effect of BclA when used as a DNA vaccine in conjunction with a DNA vaccine expressing PA (104) (see below). More recently, only a minimal effect of vaccination with BclA was shown where it prolonged time to death but did not protect mice against a low-dose challenge with an unencapsulated toxinogenic strain (105). Antigens present in bacilli other than toxin and capsule have also been shown to give modest protection in mice against a virulent encapsulated strain, although the nature of these antigens remains to be determined (106).

NEW DELIVERY SYSTEMS Intranasal Vaccination Various carriers and adjuvants have been used to enhance both systemic and mucosal immune responses after IN vaccination. These materials include a drug carrier prepared from soya

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phosphatidyl choline and sodium cholate (107), and the adjuvants cholera toxin, CpG ODN, Invaplex (an extract from Shigella), and MPLþTDM (108,109). Flick-Smith et al. (110) protected mice against a spore challenge with an unencapsulated toxinogenic strain after intramuscular (IM) or IN inoculation of PA, either encapsulated within poly-L-lactide microspheres or loosely bound to poly-L-lactide microspheres by lyophilization. Optimal antibody responses were observed with IM vaccination followed by an IM or IN boost. Mikszta et al. (84) and Huang et al. (111) reported that IN vaccination of mice and rabbits with a freeze-dried powder preparation of PA containing CpG ODN alone or with chitosan (112) protected rabbits against an aerosol spore challenge with a virulent encapsulated strain. The role of chitosan in protection was not determined. IN injection with dry powder formulations of PA containing a 10-mer capsule peptide (PA/MPL/chitosan/ peptide) or with a 10-mer capsule peptide-PA conjugate (PA/ MPL/Conj or PA/MPL/chitosan/Conj) were reported by Wimer-Mackin et al. (113) to protect rabbits against an aerosol challenge with a virulent encapsulated strain, although the experiments did not determine whether the capsule peptide gave any added protection above that observed with PA alone. Bielinska et al. (114) reported that mice injected IN with PA formulated with a nontoxic nanoemulsion mucosal adjuvant (PA/NE) had higher serum and mucosal antibody responses and toxin-neutralizing antibody titers compared with mice receiving formulations of PA with CpG ODN, MPL, or aluminum hydroxide. Although a quantitative comparison with AVA or PA adsorbed to aluminum hydroxide gel was not reported, IN vaccination of guinea pigs with PA/NE was fully protective against an ID challenge with a virulent encapsulated strain but only partially protected guinea pigs against an IN challenge, which showed increases in time to death (114). Another IN mucosal adjuvant that has been used in a PA vaccine is oxidized mannan that has been shown to induce antitoxin antibodies (115). Sloat and Cui reported that mice vaccinated IN with PA incorporated with liposome-protamine-DNA particles (116) or combined with polyriboinosinic-polyribocytidylic acid [poly(I:C)] (117) had a strong systemic and mucosal antibody response to PA that neutralized lethal toxin cytotoxicity. In additional studies, they showed that mice vaccinated IN with a gDPGA bovine serum albumin conjugate and PA combined with poly(I:C) produced both systemic and mucosal antibodies to gDPGA and to PA, which neutralized lethal toxin in vitro, while only systemic antibodies were induced after SC vaccination (118). It has also been reported that IN edema toxin (PA þ EF), like cholera toxin, can act as a mucosal adjuvant and stimulated both systemic and mucosal immune responses to PA or a coadministered antigen, ovalbumin (119).

Transcutaneous Vaccination Transcutaneous vaccination across abraded skin may offer an alternative to parenteral injection of a vaccine. PA adjuvanted with heat labile enterotoxin of E. coli and given by gauze patch to abraded skin induced comparable but slightly delayed antiPA antibody response and higher toxin-neutralizing antibody titers compared with mice injected IM with PA adsorbed to aluminum hydroxide (16,120,121). Mice exposed to a patch or to PA adsorbed to aluminum hydroxide were fully protected against challenge with an unencapsulated toxinogenic strain (120,121). IN challenge with a fully virulent encapsulated strain

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resulted in only partial protection of the transcutaneously vaccinated mice while the IM PA-vaccinated mice were not challenged (16). Topical application of a perflubron-based microemulsion that incorporated PA63 encoding plasmid to mice resulted in a weak antibody response that could be increased by a prior SC injection of PA formulated with aluminum hydroxide gel (122). Mikszta et al. (84) reported comparable antibody responses (ELISA and toxin neutralization) in mice or rabbits after ID injection of PA formulated with either aluminum hydroxide or CpG ODN using a microneedle or after transcutaneous vaccination using a microenhancer array (MEA) device, with or without prior abrasion of the skin, with animals injected IM with PA formulated with aluminum hydroxide. Additionally, rabbits were better protected against an aerosol challenge with a virulent encapsulated strain after IM and ID injection than rabbits receiving the MEA device (84). However, PA given ID without adjuvant elicited an antibody response and protection equivalent to that afforded by PA combined with CpG ODN.

LIVE ANTHRAX VACCINES Although live vaccines were initially developed for livestock, two toxinogenic, nonencapsulated B. anthracis strains, STI-1 and A16R, were developed and licensed for human use in the former Soviet Union (123) and China (124), respectively. Turnbull et al. (52) reported comparable anti-PA titers of guinea pigs inoculated with a single dose of Sterne or STI-1 spores but slightly higher anti-LF and anti-EF titers after STI-1 injection. In animal studies, live attenuated spore vaccines are often more effective than PA-based subunit vaccines. The increased protection afforded by live spore vaccines has been postulated (51,90,125) to be based upon the optimal presentation of bacterial antigens to the immune effector cells, including PA, LF, EF, and the presence of additional spore and vegetative antigens, which would result in a broader immune response that may involve both antibodies and stimulation of cellmediated immunity. A review of the safety and preventative efficacy of STI-1 in the former Soviet Union reported a low incidence of side effects and demonstrated efficacy in human field trials against cutaneous anthrax (123). Protection in animals has also been reported to be enhanced by using a nonproteolytic mutant of Sterne (126), which might be expected to result in higher amounts of intact PA or by combining filtered culture supernatants (37) or PA (127) with a spore vaccine. The inherent risk of live vaccine-induced adverse events might be reduced by using live recombinant bacterial strains that are more attenuated. This has been accomplished using aromatic amino acid–deficient B. anthracis mutants (Aro) prepared by transposon Tn916 mutagenesis of a Sterne derivative (128), by inactivating or deleting the enzymatic components of the exotoxins (96,97), as described above, or by using attenuated viral vectors (below). Live recombinant B. anthracis–based vaccines, more attenuated than the Sterne and STI-1 strains, were prepared by Barnard and Friedlander (129) by transforming nontoxinogenic, nonencapsulated B. anthracis strains DSterne (B. anthracis Sterne strain cured of pXO1) and DANR (B. anthracis Ames strain cured of both pXO1 and pXO2 plasmids) with shuttle vectors expressing different amounts of PA. Protection was found to correlate with the amount of PA expressed, as determined by antibody

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titers to PA. Similar findings were reported by Cohen et al. (130) who used the strong a-amylase promoter from B. amyloliquefaciens. Further development of this expression system was reported by Mendelson et al. (131), who protected guinea pigs with PA trypsin site (MASC-12) or chymotrypsin site (MASC-13) mutants, which are biologically inactive and might be less reactogenic. Some protection was observed at approximately one year after a single dose of the latter strain. A strain producing only an inactivated LF gave some protection that was less than that seen with the PA-producing strains. Aloni-Grinstein et al. (132) reported that about half of the guinea pigs vaccinated orally with live attenuated recombinant B. anthracis MASC-13 had PA and toxin-neutralizing antibody titers and were protected against a spore challenge. Another novel approach related to the live attenuated vaccine concept uses a killed but metabolically active unencapsulated toxinogenic strain with mutated LF and EF. This was shown to be immunogenic, but no protection studies have been reported (133). Bacteria other than B. anthracis have also been used as vectors for anthrax vaccines. B. subtilis strains engineered to secrete PA protected guinea pigs against challenge with a virulent encapsulated strain (128). In subsequent studies, PA and domains of PA were displayed on the spore coat and the vegetative cell in B. subtilis, together or separately, while developing an oral vaccine (134). The rationale for this approach is that B. subtilis is used commercially as a probiotic and has been used for delivering heterologous recombinant proteins (135). The highest antibody titers were in mice injected with B. subtilis recombinants that expressed both PA domains 1b-3 on the spore surface and full-length PA secreted by the vegetative cell. Intraperitoneal (IP) injection of spores of this recombinant B. subtilis clone protected mice against an IP spore challenge with an unencapsulated toxinogenic strain (134). PA has also been expressed in Lactobacillus casei for oral delivery, but this strain failed to induce an immune response in mice (136). An early attempt based on expressing PA in Salmonella typhimurium as an oral vaccine was met with limited success based on measurement of colonization, anti-PA titer elicited, and protection of mice from challenge (137). Improvement in the genetic stability, the serological response to PA, and survival after intravenous but not oral vaccination was observed when the gene encoding PA was fused with a fragment of the hemolysin A gene of E. coli (to improve PA export) and integrated into the chromosome of an auxotrophic mutant of Salmonella typhimurium (138). Galen et al. (139) expressed domain 4 of PA fused with the chromosomal cytolysin A (ClyA) hemolysin in Salmonella typhi to increase secretion. They reported that 11/15 (73%) mice seroconverted after IN inoculation with the live vector expressing the exported ClyAPA fusion, compared with only 1/16 vaccinated with a live vector construct in which domain 4 was expressed in the cytoplasm. Oral vaccination of mice with Salmonella typhimurium expressing ClyA fused with either full-length PA, PA domains 1 and 4, or PA domain 4 gave 83%, 25%, and 0% protection against an aerosol challenge with an unencapsulated toxinogenic strain, respectively (79). However, mice injected IM with each of the respective recombinant proteins had higher antibody titers and were fully protected against an aerosol challenge. This suggests that while vaccination with oral Salmonella vectors can be protective, further improvements in immunogenicity will be required to exploit the advantages of this oral delivery system.

GENETIC VACCINATION SYSTEMS The potential of genetic delivery methods, which include the use of viral vectors or plasmid DNA, lies in their ability to deliver multiagent or multicomponent vaccines. Serological responses to plasmid DNA vaccines, however, have been relatively weaker compared with viral vector vaccines.

Recombinant Viral Vectors The use of recombinant viral vectors as vaccines for anthrax was first reported using vaccinia virus Connaught and WR strains expressing PA that were shown to protect mice and partially protect guinea pigs from challenge with a virulent encapsulated anthrax strain (71,140). Venezuelan equine encephalitis virus propagation-deficient replicon particles expressing PA were also shown to protect mice from challenge with an unencapsulated toxinogenic strain (141). Replicationdeficient human serotype 5 adenovirus vector (Ad5) expressing PA has been reported to produce a more rapid and higher serological response and increased protection of mice against a lethal toxin challenge than a single IM dose of 25 mg of PA adsorbed to aluminum hydroxide gel (142). Replicationdeficient Ad5 has also been prepared expressing domain 4 of PA (143) and shown to protect mice from challenge with an unencapsulated toxinogenic strain when given after a priming dose of a plasmid expressing PA domain 4. Immunogenicity was highest when the priming dose was with the plasmid expressing PA rather than with the adenovirus vector expressing PA, suggesting that anti-vector immunity interfered with the immune response. To circumvent possible preexisting immunity to some adenovirus serotypes as well as an immune response against the vector after the primary injection, Hashimoto et al. (144) demonstrated that Ad5 preexisting immunity did not interfere with vaccination against a lethal toxin challenge with a nonhuman primate–derived adenovirus serotype C7 expressing PA. Mice with Ad5 preexisting immunity however, were not protected when vaccinated with an Ad5-based vector expressing PA.

Plasmid DNA Vaccines The first demonstration that an anthrax DNA vaccine could elicit antitoxin immunity was reported by Gu et al. (145) using a plasmid encoding PA63 and a human plasminogen activator signal secretion sequence. Williamson et al. (146) observed that higher antibody titers to PA required a PA protein booster after a primary inoculation with a DNA vaccine expressing fulllength PA without a secretion signal. Price et al. (147) protected mice against a lethal toxin challenge using a plasmid expressing either PA63 or LF10–254, the amino portion of LF that lacks the catalytic domain without signal secretion sequences. Coadministration of both plasmids resulted in higher serum antibody titers to both PA and LF than when administered alone, which were further increased with a booster injection of either PA or LFE687C, an enzymatically inactivated mutant. Using plasmid DNA encoding full-length PA83 with or without a signal secretion sequence, Hahn et al. (148) demonstrated protection of mice against challenge with an unencapsulated toxinogenic strain. In another study (149), albeit with small numbers of animals, rabbits injected IM with PA63 or PA63 and LF10–254 DNA vaccines with a signal secretion sequence, followed by a booster protein immunization with PA and/or LFE687C, were protected against aerosol challenge with a virulent encapsulated strain; in contrast, injection of PA63 or PA

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and LF10–254 DNA ID without protein boosts was not protective. In addition, LF10–254 DNA alone followed by a LFE687C booster was not protective (149). Using a similar plasmid expressing PA83 with a signal sequence, Riemenschneider et al. demonstrated protection of rabbits against a lethal SC virulent spore challenge (150). Subsequently, it was shown that rabbits were protected against an aerosol challenge with a virulent encapsulated strain after IM vaccination with codon-enhanced plasmid constructs expressing PA lacking the furin cleavage site (PA83D furin) and with the PA83Dfurin plasmid coadministered with plasmids expressing N-terminal fragments of LF (LF34–295 and LF34–583) formulated with cationic lipids possessing adjuvant properties without protein boosting (91). These experiments did not determine whether including the LF plasmid was of any additional benefit to the PA plasmid vaccine alone. Although plasmid construct LF34–583 given by itself did not increase overall survival, it resulted in a statistically significant delay in time to death compared with animals given a control plasmid. The results of these studies led to the testing of the DNA vaccines in phase 1 clinical trials (see below). The possibility of including a DNA vaccine for anthrax as part of a multiagent DNA vaccine has also been reported (150,151).

VACCINES IN CLINICAL TESTING Proof of Efficacy of Candidate Vaccines The licensure of currently available vaccines such as AVA was based on efficacy being inferred from a single field trial conducted in mill workers with a similar but less potent PA-based vaccine, and supported by a subsequent review conducted by the Centers for Disease Control (CDC). The field trial demonstrating efficacy included mainly cases of cutaneous and only a few cases of inhalational anthrax (47). Although it was not possible to evaluate efficacy against inhalational anthrax alone because the incidence was too low, all five cases occurred in unvaccinated workers. AVA was licensed in 1970 for preexposure vaccination to protect against all forms of anthrax disease. Further evidence for efficacy is the CDC data from 1962 to 1974 that indicated that no cases of anthrax occurred in fully immunized at-risk workers (152). An extensive analysis by the National Academy of Sciences concluded that AVA is effective for the protection of humans against anthrax, including inhalational anthrax (65). To date, no vaccine has been licensed for postexposure use, although research in nonhuman primates supports such a use to reduce the duration of antibiotic prophylaxis (22). The occurrence of anthrax, especially inhalational anthrax, is too infrequent to test vaccine efficacy in controlled clinical trials, and it would be unethical to perform human challenge studies for this potentially fatal disease. In 2002, the U.S. FDA revised its regulations and created a new regulatory mechanism, the ‘‘Animal Rule’’ to allow demonstration of efficacy of vaccines or therapeutic products in appropriate animal models for diseases, such as anthrax, where it is not possible to conduct either field trials or challenge studies in humans (153). The Animal Rule can be used as a substitute for clinical proof of efficacy, provided four conditions are met: (i) there is a reasonably well-understood pathophysiological mechanism of toxicity and its amelioration by the product; (ii) the effect (the benefit of the product) is demonstrated in more than one species, unless one animal model is characterized sufficiently to be confident that it predicts response in humans; (iii) the endpoint is clearly related to be of benefit in

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humans, usually survival or prevention of morbidity; and (iv) there is sufficient information in the animal model to allow selection of an effective dose in humans. For PA-containing vaccines, the vaccines closest to or already in clinical trials, it appears that only inhalational challenge models in rabbits and nonhuman primates would meet these criteria.

Clinical Trials of Candidate Vaccines As described above, it is anticipated that licensure of anthrax vaccines will require safety and immunogenicity to be established in large phase 2 clinical trials, whereas the equivalent of phase 3 or pivotal efficacy trials will have to be undertaken as appropriate inhalational challenge studies in rabbits and nonhuman primates. In these studies, the candidate vaccine will have to demonstrate protection with doses of vaccine that induce levels of an as yet to be defined serological marker that correlates with immunity, likely functional antibody such as TNA (see earlier text), no higher than the levels that can be achieved in humans with the vaccine. A critical factor in such studies is the development of a correlate of immunity in animals that can be extrapolated for use in humans. Confirmation of efficacy by passive protection in the animal models, using plasma obtained from vaccinees in clinical trials may also be required. For postexposure prophylaxis (PEP), it is likely that vaccine will always be given in conjunction with antibiotics. For this reason, the models will require an antibioticalone control group, and proof of vaccine efficacy in the PEP model will require demonstration that animals receiving both vaccine and antibiotics postexposure have improved survival compared with those receiving antibiotics alone. Some data supporting the value of PEP vaccination when added to antibiotics have been reported in the nonhuman primate model of inhalational anthrax (22). Anti-PA IgG measured by ELISA or in a functional assay, TNA, appears to be a correlate of protection in animal studies as described above, and qualified assays have been developed (33,154) that can be used to bridge animal challenge studies with immunogenicity achievable in humans. While there appears to be good linear correlation between TNA and ELISA for both AVA and recombinant PA (rPA) vaccines, the correlations differ between the two vaccines so that TNA and ELISA results may not be interchangeable (155,156). In one study, it appeared that for any given level of TNA, the corresponding level of anti-PA IgG assayed by ELISA was higher with AVA than rPA, suggesting that AVA may be inducing additional nonneutralizing antibodies (155). The only anthrax vaccines currently in clinical trials are PA-based vaccines, as summarized in Table 2. These include AVA, ‘‘second-generation’’ rPA vaccines, and a bivalent DNA vaccine encoding genes for PA and LF. In AVA, the PA is mixed with aluminum hydroxide adjuvant, though it is possible that the formalin in the vaccine may also increase immunogenicity (157). The effects of giving a toll-like receptor 9 (TLR-9) agonist, CpG 7909, along with AVA administered IM was investigated in a phase 1 study (158). The CpG plus AVA group had peak TNA titers that increased almost nine-fold when compared with AVA alone. However, grade 2 and 3 adverse events—fatigue, headache, muscle aches, pain, and limitation of arm motion—were substantially increased in the CpG/AVA group compared with the AVA alone group (158). Whether CpG or other TLR-9 agonists would have given a similar response when administered with PA

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Table 2 Recent Clinical Trials With Anthrax Vaccines Vaccine

Technology

Adjuvant

AVA

Filtered culture supernatant of V770-NP1-R strain of B. anthracis

Al(OH)3 (600 mg as Phase 4 studies to modify route (SC to Al3þ); other IM) and immunization B. anthracis proteins schedule and number and formalin may of doses contribute to adjuvant effect

RPA

Recombinant PA purified from sporulation-deficient, nontoxinogenic B. anthracis

Recombinant PA purified from E. coli Bivalent Genes for inactive PA plasmid and lethal factor (LF) DNA

Type of clinical trial

CpG in addition to current adjuvant

Phase 1 study of tolllike receptor 9 agonist (CpG 7909) combined with AVA, administered IM

Alhydrogel (82.5 mg as Al3þ)

Phase 1 ascending dose tolerance of IM rPA

Alhydrogel (800 mg as Al3þ) ‘‘mixed at bedside’’ or no adjuvant

Phase 1 ascending dose tolerance study of IM rPA

Alhydrogel (dose not disclosed)

Phase 1 ascending dose tolerance study of IM rPA Phase 1 ascending dose tolerance study

Outcome

Reference

IM less reactive than SC; peak antibody after IM less than SC at 8 weeks, but ‘‘noninferior’’ at 7 mo The number of doses administered may be decreased: dosing at two weeks might be omitted for pre-exposure immunization Local and systemic reactions increased in AVA þ CpG compared with IM AVA. Immunogenicity increased 9-fold compared with IM AVA

159, 160

All doses well tolerated; local reactogenicity lower compared with IM AVA; systemic reactogenicity higher compared with IM AVA. Peak antibody after highest rPA dose (75 mg) lower, but noninferior, compared with IM AVA All doses well tolerated; reactogenicity similar to AVA. Peak antibody to adjuvanted rPA equivalent to SC or IM AVA. Antibody response to unadjuvanted rPA ‘‘poor’’ and inferior to adjuvanted rPA All doses well tolerated. Peak antibody responses less than IM AVA

155

Dose-limiting grade 3 toxicity. No detectable TNA response; antiPA IgG detected by ELISA in less than half of the vaccinees

158

161

162

163

Abbreviations: IN, intranasal; SC, subcutaneous; IM, intramuscular; IP, intraperitoneal; AVA, anthrax vaccine adsorbed; PA, protective antigen; TFN, toxin neutralizing antibody.

without aluminum adjuvant cannot be determined from this study, as there was not a group combining rPA without aluminum but with CpG to allow determination of the contribution of each adjuvant. In rabbits, rPA given IM or ID by itself is immunogenic, and no increase was observed when it was given with CpG (84). The schedule and route of AVA—administered SC in six injections (0, 2, 4 weeks; 6, 12, 18 months) over 18 months— were largely determined empirically. Accordingly, there have been ongoing clinical studies to determine whether AVA can be administered IM and with fewer injections. A pilot study (159) suggested that the injection at two weeks could be omitted and that IM administration might be preferable. AVA given IM at zero and four weeks was less reactogenic and elicited a peak antibody response comparable to three AVA vaccinations according to the licensed route and schedule. A larger congressionally mandated clinical study directed by the CDC is further investigating whether the schedule of AVA can be simplified and whether its route can be changed from SC to IM (160). Preliminary results from that study reported that the geometric mean anti-PA concentrations at eight weeks were lower in the groups that received IM vaccinations, either at zero and four weeks or zero, two, and four weeks, compared with the SC

group that received the zero-, two-, and four-week schedule while at seven months, both IM groups were ‘‘noninferior’’ to the SC group. Second-generation, rPA vaccines, with the rPA purified from B. anthracis (rPA102) (155,161) or E. coli (162) and mixed with aluminum (Alhydrogel) adjuvant have been studied in phase 1 ascending dosage tolerance studies. rPA102 was initially studied in a phase 1 ascending dose tolerance study where rPA doses of 5, 25, 50, and 75 mg, each formulated in Alhydrogel (82.5 mg as aluminum/dose) were administered IM at zero, four, and eight weeks (155). In this study, AVA administered IM at zero and four weeks was used as a comparator. All doses of rPA102 (and AVA) were well tolerated, without dose-limiting toxicity, although AVA appeared to have more local reactogenicity and rPA appeared to have more systemic reactogenicity (but only after the first dose). There was a dose-response relationship from 5 to 75 mg of rPA102. When TNA levels were compared, after the second dose of AVA or 75 mg of rPA102, the AVA response was higher but not significantly different (155). A phase 2 study of rPA102, which investigated higher doses of rPA and Alhydrogel, was confounded by instability of the vaccine and adjuvant, and current development has been focused on formulation

Chapter 77: Anthrax Vaccines

improvement. In a separate phase 1 study, rPA 102 expressed in B. anthracis was not formulated but mixed with Alhydrogel just prior to administration ‘‘at the bedside’’ (161). In that study, doses of rPA ranging from 5 to 75 mg given IM were similar in immunogenicity and tolerability to IM AVA, and less reactogenic than SC AVA. Immunogenicity with all doses of rPA and AVA given IM was not statistically different from AVA given SC. rPA with adjuvant was significantly more immunogenic than rPA without adjuvant (161). In a similar phase 1 study, doses of 5, 25, 50, and 100 mg of rPA produced from E. coli and adsorbed to Alhydrogel were administered IM on either a 0- and 21- or 0- and 28- day schedule, with an AVA comparator given IM at 0 and 28 days. Reactogenicity was low and not dose limiting. A dose response in antibody to PA was noted from 5 to 50 mg rPA but not between 50 and 100 mg. The peak antibody response was statistically inferior to the peak after AVA, and it appeared that a third injection might be required for primary vaccination (162). All three studies of second-generation rPA vaccines have used early formulations. It does not appear that the optimized dose and formulation of an rPA vaccine have as yet been determined or evaluated in clinical trials. A cationic lipid-formulated bivalent plasmid DNA-based vaccine, which encoded biologically inactive PA and LF genes lacking the furin cleavage site and the metalloprotease domain, respectively, was studied in a small phase 1 ascending dose tolerance study (163). The vaccine protected rabbits in an inhalational challenge model (91) (see above). However, the initial results in humans were disappointing. An IgG anti-PA response as measured by ELISA was detected in less than half the participants; there was dose-limiting grade 3 local and systemic toxicity, and there was no detectable functional antibody response by TNA in any participant.

SUMMARY The currently licensed anthrax vaccine is safe and effective, but studies in recent years have focused on improving the immune response particularly by reducing the number of doses. The major immunogen in the licensed and new vaccines in clinical trials is PA. Advances can be expected in several areas including the use of new adjuvants and transcutaneous, respiratory, and genetic delivery systems to improve the rapidity and extent of the immune response. Future improvements in vaccines may also include additional antigens that may enhance the efficacy of PA-based vaccines. These approaches include incorporating the other toxin proteins, the capsule, spore antigens, and the use of live attenuated vaccines to generate multicomponent vaccines that may elicit more protective and rapid immune responses.

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133. Skoble J, Beaber JW, Schnupf P, et al. Development of a killed but metabolically active anthrax vaccine. Bacillus ACT 2005 September 25–29, 2005 Santa Fe, NM (abstr p 40, 55). 134. Duc LH, Hong HA, Atkins HS, et al. Immunization against anthrax using Bacillus subtilis spores expressing the anthrax protective antigen. Vaccine 2007; 25:346–355. 135. Duc le H, Hong HA, Fairweather N, et al. Bacterial spores as vaccine vehicles. Infect Immun 2003; 71:2810–2818. 136. Zegers ND, Kluter E, van Der Stap H, et al. Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: towards the development of an oral vaccine against anthrax. J Appl Microbiol 1999; 87:309–314. 137. Coulson NM, Fulop M, Titball RW. Bacillus anthracis protective antigen, expressed in Salmonella typhimurium SL3261, affords protection against anthrax spore challenge. Vaccine 1994; 12:1395–1401. 138. Garmory HS, Titball RW, Griffin KF, et al. Salmonella enterica serovar Typhimurium expressing a chromosomally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge. Infect Immun 2003; 71:3831–3836. 139. Galen JE, Zhao L, Chinchilla M, et al. Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA. Infect Immun 2004; 72:7096–7106. 140. Iacono-Connors LC, Welkos SL, Ivins BE, et al. Protection against anthrax with recombinant virus-expressed protective antigen in experimental animals. Infect Immun 1991; 59:1961–1965. 141. Lee JS, Hadjipanayis AG, Welkos SL. Venezuelan equine encephalitis virus-vectored vaccines protect mice against anthrax spore challenge. Infect Immun 2003; 71:1491–1496. 142. Tan Y, Hackett NR, Boyer JL, et al. Protective immunity evoked against anthrax lethal toxin after a single intramuscular administration of an adenovirus-based vaccine encoding humanized protective antigen. Hum Gene Ther 2003; 14:1673–1682. 143. McConnell MJ, Hanna PC, Imperiale MJ. Adenovirus-based prime-boost immunization for rapid vaccination against anthrax. Mol Ther 2007; 15:203–210. 144. Hashimoto M, Boyer JL, Hackett NR, et al. Induction of protective immunity to anthrax lethal toxin with a nonhuman primate adenovirus-based vaccine in the presence of preexisting antihuman adenovirus immunity. Infect Immun 2005; 73:6885–6891. 145. Gu M-L, Leppla SH, Klinman DM, et al. Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen. Vaccine 1999; 17:340–344. 146. Williamson ED, Beedham RJ, Bennett AM, et al. Presentation of protective antigen to the mouse immune system: immune sequelae. J Appl Microbiol 1999; 87:315–317. 147. Price BM, Liner AL, Park S, et al. Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect Immun 2001; 69:4509–4515. 148. Hahn UK, Alex M, Czerny CP, et al. Protection of mice against challenge with Bacillus anthracis STI spores after DNA vaccination. Int J Med Microbiol 2004; 294:35–44. 149. Galloway D, Liner A, Legutki J, et al. Genetic immunization against anthrax. Vaccine 2004; 22:1604–1608. 150. Riemenschneider J, Garrison A, Geisbert J, et al. Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine 2003; 21:4071–4080. 151. Williamson ED, Bennett AM, Perkins SD, et al. Co-immunisation with a plasmid DNA cocktail primes mice against anthrax and plague. Vaccine 2002; 20:2933–2941. 152. FDA (Food and Drug Administration). Biological products: bacterial vaccines and toxoids: implementation of efficacy review. Proposed rule. Fed Reg 1985;50:51002–51117. 153. Department of Health and Human Services, Food and Drug Administration. New Drugs and Biological Drug Products; evidence needed to demonstrate effectiveness of new drugs when

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154.

155.

156.

157.

158.

159.

human efficacy studies are not ethical or feasible. 21 CFR Parts 314 and 601. Fed Reg 2002; 67:37988–37998. Quinn CP, Semenova VA, Elie CM, et al. Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. Emerg Infect Dis 2002; 8:1103–1110. Gorse GJ, Keitel W, Keyserling H, et al. Immunogenicity and tolerance of ascending doses of a recombinant protective antigen (rPA102) anthrax vaccine: A randomized, double-blinded, controlled, multicenter trial. Vaccine 2006; 24:5950–5959. Parreiras PM, Sirota L, Wagner L, et al. Agreement between estimates of antibodies induced by anthrax vaccines containing protective antigen, using ELISA and toxin neutralization. 2006 ASM Biodefense Research Meeting February 15–18, 2006, Washington, D.C. (abstr 201, p 67). Little SF, Ivins BE, Webster WM, et al. Effect of aluminum hydroxide adjuvant and formaldehyde in the formulation of rPA anthrax vaccine. Vaccine 2007; 25:2771–2777. Rynkiewicz D, Rathkopf M, Ransom J, et al. Marked enhancement of antibody response to anthrax vaccine adsorbed with CPG 7909 in healthy volunteers. 45th Annual ICAAC Meeting December 16–19, 2005 (abstr LB 25). Pittman PR, Kim-Ahn G, Pifat DY, et al. Anthrax vaccine: immunogenicity and safety of a dose-reduction, route-change comparison study in humans. Vaccine 2002; 20:1412–1420.

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160. Marano N, Plikaytis BD, Martin SW, et al. Interim analysis of a human dose and schedule optimization study of the approved US anthrax vaccine adsorbed (AVA). The Tenth Annual Conference on Vaccine Research April 30–May 2, 2007, Baltimore, MD (abstr S21, p 61). 161. Campbell JD, Clement KH, Wasserman SS, et al. Safety, reactogenicity and immunogenicity of a recombinant protective antigen anthrax vaccine given to healthy adults. Hum Vaccin 2007; 3:205–211. 162. Dyson EH, Irving MS, Goldwater R, et al. An ascending dose study to assess the safety and tolerability of an rPA anthrax vaccine, and compare its immunogenicity with that of Anthrax Vaccine Adsorbed (AVA). 46th Annual ICAAC Meeting September 27–30, 2006, San Francisco, CA (abstr G¼156c). 163. Keitel WA, Treanor J, El-Sahly H, et al. A phase I, double-blind, dosage-escalating clinical trial to evaluate the safety and immunogenicity of prophylactic anthrax DNA vaccine in healthy adults. 43rd Annual Meeting of the Infectious Diseases Society of America October 6–9, 2005, San Francisco, CA (abstr 1022, p 220).

Disclaimer: Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.

78 Tularemia Vaccines Alan S. Cross and Eileen M. Barry Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

HISTORY/EPIDEMIOLOGY/CLINICAL Tularemia, a zoonotic infection caused by Francisella tularensis, is an infection of the northern hemisphere with highly localized endemic foci. It was first reported by McCoy in 1911 as a disease of ground squirrels dying of a plague-like disease in Tulare County, California. The first reported human cases of tularemia were reported in 1914. Clinical disease most commonly manifests as a cutaneous infection in individuals exposed to infected animals or bitten by arthropods (e.g., ticks) that carry the bacteria. In 1942, Francis estimated that two-thirds of all American cases of tularemia were linked to contacts with cottontail rabbits (1). It has been stated that ‘‘. . .no other infection of animals communicable to man . . . can be acquired from sources so numerous and so diverse’’ (2). While other routes of infection are more common, inhalational tularemia results in significant morbidity and occasional mortality. Before antibiotics, systemic infection with type A strains acquired following inhalation had a mortality rate of 30% to 60% (3). Although well-described sporadic cases have been acquired from the environment (e.g., gardeners at Martha’s vineyard), widespread pulmonary tularemia is most likely thought to be acquired following its intentional release during a biological attack. In 1969, the World Health Organization estimated that aerosolization of 50 kg of F. tularensis in area of 5 million people would incapacitate 250,000 and kill 19,000 individuals (4). Using these data, the U.S. Centers for Disease Control and Prevention (CDC) concluded that such an attack would cost $5.4 billion/100,000 individuals affected (5). Such a high cost and morbidity led the CDC to designate F. tularensis as one of six category A select agents.

Bacteriology Francis originally named the organism Bacterium tularensis in 1919; it was later designated Pasteurella tularensis, and finally renamed Francisella tularensis in 1947 (6). Subsequent sequence analysis of 16S rDNA resulted in its placement in Proteobacteria. With its high cell wall lipid content and unique cellular fatty acid composition, Francisella is the only recognized genus in the family Francisellaceae. There are four subspecies. F. tularensis subsp. (ssp.) tularensis (type A), the most virulent subspecies, is found only in North America. As few as 10 bacteria of F. tularensis ssp. tularensis can cause subcutaneous infection in man, while 25 organisms can do so by the aerosol route (7). A less virulent strain, F. tularensis ssp. holarctica (type B), is found in North America, Europe, and Asia, while strains from Central

Asia have been designated F. tularensis ssp. mediasiatica. A fourth subspecies, F. tularensis subsp novicida is not considered a human pathogen.

Virulence The manifestations of the disease are most likely associated with the host cellular inflammatory response induced by F. tularensis infection (8). Not only can F. tularensis infect phagocytes (macrophages, neutrophils, and dendritic cells), but also nonprofessional phagocytes such as hepatocytes, endothelial cells, and alveolar type II cells. Since F. tularensis is considered an intracellular organism, most of the work on its virulence has been conducted in macrophage cell cultures. F. tularensis does not produce any obvious exotoxins, and its lipopolysaccharide (LPS) is not endotoxic. One Francisella pathogenicity island (FPI) has been identified, which contains genes iglA-D, pdpA, and pdpD that encode proteins whose expression is regulated by macrophage growth locus genes, mglA and mglB. Within the macrophage, F. tularensis blocks phagosome maturation and acidification, and disrupts the phagosomal membrane which permits escape of F. tularensis into the cytoplasm where intracellular growth leads to both activation of caspase pathways and apoptosis (9). To exert its virulence both in vitro and in vivo, F. tularensis must escape from the phagosome, an event that depends on the expression of a pathogenspecific 23-kDa protein, encoded by intracellular growth locus gene, iglC, also located on the pathogenicity island. While its function is unknown, iglC loss results in complete loss of virulence as well as an inability to induce a protective immune response, perhaps through an inability to allow secreted proteins to enter the histocompatibility complex (MHC) class I pathway. It has been observed that iglC mutants of F. tularensis remain within the phagosome, where they are still able to induce a robust TLR2 response, but are unable to induce apoptosis (10). Genes that do not appear to influence the behavior of F. tularensis in macrophages, such as a putative type IV pilin gene or tolC, may also play a role in virulence (11,12). Type IV pili are virulence factors for many bacteria. Genes encoding the type IV pili were identified in the ssp. tularensis strain, Schu S4 genome sequence, but its precise role in infection is not known. Type IV pili appear to be involved in the dissemination of ssp. holarctica from its initial site of infection. The O-polysaccharide (PS) of the LPS, which is common to ssp. tularensis and holarctica, is essential for virulence. The

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O antigen limits complement protein C3 deposition on the surface of types A and B F tularensis and enables them to resist complement-mediated lysis (13). Type B (live vaccine strain, LVS) and type A (Schu S4) strains differ in virulence. One potential mechanism of Schu S4 virulence is the active suppression of the pulmonary immune response, in part through the induction of TGF-b, which allows Schu S4 to evade detection and to actively suppress the in vivo responses to secondary stimuli, such as LPS (14). Undoubtedly, additional virulence genes will be identified in Schu S4. In animal studies, the host response is a major factor in the morbidity and mortality of tularemia. Neutrophils appear to play a role in host defenses against F. tularensis infection inasmuch as mice unable to recruit these cells into infectious foci rapidly die from lower doses of LVS (15). Francisella tularensis also blocks the respiratory burst within polymorphonuclear leukocytes (PMNs) (16). Matrix metalloprotein 9, a neutrophilic protein, increases host susceptibility and lack of this protein renders mice protected against Schu S4 (17). Infection with F. tularensis ssp. holarctica induces massive expansion of circulating gD T cells whose function is unknown (18).

MECHANISMS OF HOST PROTECTION Antibodies In mice, antibodies alone can protect against lethal systemic challenge with low virulence strains, but not against aerosol challenge with a fully virulent type B strain or against either systemic or aerosol challenge with type A strains. Human subjects immunized with the LVS mount an antibody response to the LVS LPS and to many protein antigens, although no correlation was found between levels of agglutinating antibody to Foshay or LVS vaccines and protection. Passive immunization with serum collected from mice immunized with either a heat-killed preparation of F. tularensis LVS or an O-antigen deficient mutant yielded similar protection against homologous live LVS challenge. These data suggest that antibodies alone can confer protection against LVS challenge, and that these protective antibodies are not dependent on anti-O-specific antibodies (19). The protective role of serum antibodies against Francisella tularensis was also demonstrated when immune serum was passively administered to naı¨ve mice before respiratory challenge with LVS. The protective effect of this serum prophylaxis (100%) was independent of complement, but required interferon gamma (IFN-g). Since severe combined immunodeficiency (SCID) mice were not protected by passive antibody transfer, cooperation between humoral and cellular immune responses was considered necessary for sterilizing immunity to F. tularensis, and that T cell, not NK cells, might be the source of this IFN-g (20). Protection against respiratory LVS tularemia by intranasal administration of inactivated F. tularensis LVS required exogenous IL-12 as an adjuvant. Interestingly, mice genetically deficient in immunoglobulin A expression did not survive. Thus, IgA-mediated protection may have a role in protection against pulmonary tularemia following mucosal immunization (21).

Adaptive Immune Response Given the relatively recent development in our understanding of the adaptive immune response, assays of cellular immune responses were not performed in early human vaccine studies.

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The Soviets considered the duration of immunity with their vaccines to be five years, although more recent data with the LVS suggest that cell-mediated immunity to LVS persists for at least 25 years (22). While it is assumed that the adaptive immune responses elicited by LVS are primarily responsible for the protection, there is little direct evidence. It has been shown that long-lasting specific CD4þ and CD8þ T-cell immunity and proliferation of natural killer (NK) and monocytes developed against protein antigens after LVS immunization. Further, protection afforded to mice by LVS against type A strains can be abrogated by depletion of either CD4þ, CD8þ T cells or by neutralization of IFN-g prior to challenge (23). This remains the best evidence of cellular immune response as being required for efficacy of LVS in mice; however, one cannot disregard the possibilities that antibodies to other (protein) antigens may play a role.

VACCINES While the relatively limited burden of naturally acquired human tularemia disease would not argue for the need for a vaccine, the previous use and deployment of F. tularensis as a bioweapon in the 1940s (Soviets, Americans, and Japanese in Unit 731 before World War II) has led to the development of vaccines to provide a countermeasure. During World War II, tens of thousands of soldiers on the eastern front were infected with tularemia, perhaps through its intentional release. While Soviet soldiers were said to receive mass aerosol inoculations, the major impetus for vaccine development in the United States was to protect laboratory workers engaged in biological warfare programs. Although Francis himself developed infection at least on three occasions, tularemia infection in man is generally considered to protect against subsequent infection. In experimental systems, protection is afforded by natural infection or by live attenuated strains, but little by killed vaccines (24).

Killed Vaccine The initial vaccines developed in the West were heat- and formalin-killed, but these were highly reactogenic and poorly immunogenic, perhaps due to alteration of the F. tularensis antigens. Dr Lee Foshay prepared a killed whole-cell vaccine by acid/phenol extraction that was not highly efficacious against challenge of mice and nonhuman primates with Schu S4, a protype type A strain; however, studies in humans demonstrated that it both reduced the number of infections and if infected, modified the course of disease (25). This vaccine was administered to several thousand individuals, including laboratory workers at high risk of infection, and was better tolerated than previous killed vaccines (26).

Live Attenuated Vaccine The Foshay vaccine modulated ulceroglandular and typhoidal tularemia, but did not protect against type A infection. The failure of killed vaccines to induce solid protection has been attributed to its failure to induce a potent cellular immune response, which is considered necessary for protection against virulent type A infection. The only vaccines in wide use against bacteria that like F. tularensis are intracellular are live attenuated organisms. These include BCG (Bacillus Calmette-Gue´rin) against tuberculosis and strain Ty21 and live oral vaccine against typhoid fever; both these live vaccines induce potent cellular immune responses (8).

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LVS Vaccine The development of live attenuated vaccines by Soviet scientists in the 1930s and 1940s led to human testing in 1942. The vaccine protected against virulent challenge at six months after immunization. Approximately 60 million Soviet citizens were immunized with the live attenuated vaccines between 1946 and 1960. However, since no type A strains are found in the USSR, its efficacy could be shown only against type B F. tularensis. In one of the more remarkable episodes of Soviet-American cooperation during the height of the Cold War, under a formal scientific exchange in 1956, scientists at the Gamla Institute in Moscow provided one of their vaccine strains, designated strain 15, to U.S. scientists. Eigelsbach and colleagues passaged the Russian strain and recognized two variant colony morphologies, one blue and the other gray when viewed under oblique light. The blue variant had an LD50 in mice of 106 CFU following IP administration while the gray variant had an LD50 1000-fold higher. Immunization of mice subcutaneously with 102 to 106 CFU blue variants protected against subsequent subcutaneous challenge with 103 CFU of the Schu S4 strain, while all mice similarly immunized with 108 CFU of the gray variant died after challenge. This blue variant underwent further passage and was designated the LVS (78). This ‘‘pragmatically attenuated’’ strain (i.e., not attenuated with a defined mutation) of F. tularensis ssp. holarctica (LVS) soon supplanted use of the Foshay killed vaccine for immunization of laboratory workers at Ft. Detrick. Subsequent retrospective, longitudinal studies of these laboratory workers in the tularemia program established that immunization with either killed or LVS vaccines resulted in a lower incidence of laboratory-acquired tularemia (the incidence of respiratory tularemia decreased dramatically) compared to unimmunized workers, and that receipt of the LVS vaccine resulted in a more benign course if infected than those receiving the Foshay strain (27). LVS reduced the incidence of laboratory-acquired tularemia from 5.7 to 0.27 cases per 1000 at-risk employee years; however, the incidence of ulceroglandular tularemia in this group was similar with either vaccine, though less severe in the LVSimmunized group (27). Since it was impossible to establish the time of diseasecausing exposure of laboratory workers to F. tularensis, it was difficult to determine a precise assessment of the efficacy of these vaccines through a retrospective review. Consequently, Woodward and colleagues developed a human challenge model, which was justified on the basis of (i) no animal model of tularemia faithfully duplicated the human infection and (ii) administration of streptomycin at the onset of fever quickly aborted the infection (28). Following multiple challenge studies in monkeys, the LVS vaccine was tested for efficacy in human subjects (conscientious objectors, prisoners, and army recruits) under a U.S. Army program, ‘‘Operation Whitecoat.’’ A series of challenge studies was conducted in subjects who were immunized with the LVS vaccine by various routes, and then challenged with the either the non-attenuated type B strain or the fully virulent Schu S4 type A strain. These studies confirmed that LVS vaccination conferred at least partial protection against challenge with Schu S4. The degree of protection induced by scarification immunization with LVS against aerosol challenge with wild-type organisms was related to the level of challenge dose, and was superior to the Foshay-type killed vaccine (29). In an attempt to increase the protective capacity of LVS vaccination, Hornick

and colleagues (30) administered LVS by the aerosol route to a total of 253 volunteers. At the highest dose (108 CFU), the volunteers experienced mild typhoidal tularemia lasting 2.5 days. Volunteers immunized by aerosol with a high dose of LVS or a low dose of LVS exhibited 100% and 50% to 73% protection, respectively, when challenged with 2.5  104 CFU of aerosolized Schu S4 (2500 times the minimum infective dose). Volunteers vaccinated with LVS by scarification exhibited 54% protection against disease following this challenge (30). Since the LVS was more virulent for humans when given by aerosol, it was subsequently administered only by scarification. In sum, these studies demonstrate that vaccination with well tolerated doses of LVS either by scarification or aerosol delivery conferred only partial protection against challenge with virulent type A F. tularensis delivered by aerosol or intradermally. Immunization of laboratory workers at Ft. Detrick with LVS later demonstrated that this organism is detectable by PCR at the inoculation site in over half of subjects for seven days and then disappears. No blood samples were positive (31). Drawbacks of the LVS vaccine. The LVS vaccine for tularemia has been approved under an investigational new drug (IND) by the FDA, but is not licensed. LVS suffers from several drawbacks that make it a suboptimal vaccine. LVS is based on a type B strain. It is widely believed that a vaccine based on a type A strain will confer superior protection against type A challenge. Supporting this hypothesis is a study in which rabbits vaccinated by the subcutaneous route with Schu S4, followed by streptomycin treatment to prevent mortality, were protected against a subcutaneous challenge with Schu S4 (103 CFU) whereas animals immunized with LVS were not protected (32). Despite the sequencing of the complete genomes of LVS, Schu S4, and five other Francisella strains, the mechanism of attenuation in LVS has not been completely defined. Multiple genetic polymorphisms were identified in LVS compared to other type B strains, including clinical isolates, and subsequent proteomic studies suggested candidate genetic differences that may account for attenuation (33,34). The contributions of each of these defects to the overall attenuation of LVS are unknown, as is the capacity for reversion to virulence, the latter being an important concern considering the potential mass use of this vaccine. Further, inconsistencies in the LVS phenotype, manifested as blue or gray colony variants, have been observed. The variants have different virulence characteristics and protective capacities when tested in animal models (35). Although not formally demonstrated to be responsible for the observed phenotype, the gray variants were found to lack LPS O-antigen (36). The instability of LVS following growth in different media is also problematic for its manufacture at an industrial level. Finally, there are no easily measured correlates of protection for LVS (or for that matter, any attenuated vaccine strain) that can demonstrate the level of protection if one were exposed to virulent F. tularensis. New lot of the LVS vaccine. Given the safety record of the LVS vaccine and its ability to protect at least partially against an aerosol type A challenge, a new lot of vaccine was produced. Preliminary studies have demonstrated the safety and immunogenicity of the improved lot in the rabbit model (37), and clinical trials are under way. Rationally-Attenuated Live Vaccines Following the events of September 2001, there was concern that bioterrorist attacks with the intentional release of select agents

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Table 1 Rationally mutated strains of F. tularensis as vaccine candidates Strain background Gene LVS LVS LVS LVS LVS Schu Schu Schu Schu

S4 S4 S4 S4

purMCD guaB, guaA sodB wbt1 wbtA purMCD dsbB FTT1103 FTT0198

Function

Virulence and protective capacity in the mouse model

References

Purine biosynthesis Purine biosynthesis Superoxide dismutase O-antigen synthesis O-antigen biosynthesis Purine biosynthesis Disulfide bond formation Lipoprotein 58-kDa protein

Attenuated, protective against LVS Attenuated, protective against LVS Attenuated, partial protection against Type A Attenuated, partial protection against LVS Attenuated and protective against LVS Attenuated, partial protection against Type A Attenuated, not protective against Type A Attenuated, protective against Type A Attenuated, partial protection against Type A

46 47 48 49 50 51 52 53 54

Abbreviation: LVS, live vaccine strain.

might soon follow. As a result, further work attempted to fully characterize the LVS strain, and the development of new vaccines based on type A and type B strains for tularemia achieved a new urgency. Prior to 2001, only a few investigators studied the pathogenic mechanisms of F. tularensis (38–41). Consequently, few techniques for its genetic manipulation were available. Reports of transformation and electroporation methods allowed numerous groups to attempt allelic exchanges for the generation of targeted attenuation mutations (42–44) Subsequently, multiple reports on the manipulation of Francisella subspecies holarctica and novacida identified genes required for pathogenesis and revealed virulence mechanisms (45). Many of these rationally mutated strains of F. tularensis have been tested as experimental vaccines in animal models (Table 1). Attenuation of LVS. The O-PS of the F. tularensis LPS is identical in type A and B strains, and all Francisellae share a common core oligosaccharide and lipid A (55,56). The role of this antigen has been explored in relation to vaccines. The O-PS of F. tularensis provides good protection against systemic challenge with attenuated and virulent type B strains, but not against systemic type A or aerosol challenge with type A or B F. tularensis. Thus, an LPS-based vaccine could combat natural type B infections from insect bites of the skin or contact with contaminated animals. Two groups induced mutations in LVS with modified expression of O-PS antigen (49,50). An LVS-specific gene with homology to a galactosyl transferase was selected for allelic replacement. One of these resulting mutants, WbtI (G191V), was serum-sensitive and highly attenuated in mice. Immunization with this strain protected against a relatively low-dose IP challenge with LVS, but was less protective against a high-dose challenge (49). Inactivation of a wbtA-encoded dehydratase of the O antigen PS locus of LVS resulted in loss of virulence with a complete lack of surface O-PS expression. This serum sensitive strain conferred protective immunity in mice against challenge with an otherwise lethal dose of LVS or fully virulent type B strain (50). Attenuation of Schu S4. While suicide plasmid systems, transposon mutagenesis and other molecular genetic techniques are now considered routine for use in F. tularensis holarctica and novacida, and several mutated LVS derivatives have been characterized, only a few publications have appeared on the genetic manipulation of type A strains (Table 1) (43,52,54,55,57). Since genomic analysis has revealed differences between types A and B Francisella strains at the genomic level, an attenuated type A strain may offer better protection against virulent type A strains, particularly if there are differences in the protective antigens required. For example, due to a

spontaneous deletion, LVS lacks type IV pili, which may be important for attachment to the surface of a targeted cell. An isogenic mutant of F. tularensis ssp. holarctica unable to express PilA was also attenuated in virulence. Development of rationally attenuated, defined mutants may involve targeting either biosynthetic pathways (e.g., purine biosynthesis) or virulence factors (51,58). Sjostedt’s group was the first to construct a defined mutation in Schu S4 in the FTT0918 locus, encoding a 58-kDa outer membrane protein (OMP)(54). This strain was attenuated for virulence in the mouse model, and able to protect against ID challenge with wild-type type A F. tularensis. This attenuated Schu S4 mutant strain was safer than LVS and more protective against aerosol challenge with a type A strain. Recently, Zahrt and colleagues reported the construction of a purMCD mutant of Schu S4 that was avirulent in mice and, when delivered intranasally, provided limited protection against intranasal wild-type challenge that was similar to the protection afforded by LVS (51). Interestingly, intranasal vaccination with the Schu S4 DpurMCD strain alone resulted in multiple small foci of acute inflammation in the lung. This observation led the investigators to hypothesize that the foci of damage could be detrimental to proper lung immunity in response to Schu S4 challenge (51). These authors suggested that a live attenuated vaccine based on a wild-type type B strain may provide protection without the concomitant tissue damage observed with the type A mutant. Along these lines, Bakshi and colleagues demonstrated that compared to the LVS strain, a sodB mutant of LVS that is hypersensitive to oxidative stress induced a higher level of protection and reduced bacterial burden against intranasal Schu S4 challenge in the mouse model (48). Qin, Scott and Mann constructed a dsbB mutant derivative of Schu S4 that was attenuated for virulence in mice but unable to protect against challenge with the wild-type strain (52). These investigators have recently reported the development of a FTT1103 mutant derivative of Schu S4 that is both attenuated for virulence in the mouse model and able to protect against intranasal challenge with wild-type F. tularensis (53). FTT1103 encodes a protein with homology to DsbA-like proteins. This is the first report of a live attenuated Francisella vaccine that can protect against an intranasal challenge containing 1000 CFU of a type A strain in both BALB/c and the more stringent C57BL/6 mouse models.

Advantages and disadvantages of live attenuated vaccines.

With live attenuated vaccines, there is no need to identify protective antigens, as is the case with subunit vaccines (see below). They can elicit antibodies and cellular immune responses to LPS and other bacterial antigens without the need

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for external adjuvants. Challenges for development of live attenuated vaccines include achieving the correct balance between safety, a primary requirement, and immunogenicity with protective capacity. Such a vaccine must be confirmed for an inability for reversion to virulence. This can be achieved with the inclusion of two or more independently attenuating mutations.

Other Vaccine Strategies Bacterial live vectors can be used for the delivery of heterologous antigens. For example, expression of the Francisella lipoprotein antigen TUL4 in a live attenuated Salmonella vector was shown to provide partial protection against live LVS challenge (59). Jia et al. demonstrated protection against lethal type A challenge following immunization of mice with recombinant Listeria monocytogenes expressing IglC (60). Interestingly, the level of protection was less than that following LVS immunization. Other groups are pursuing a more typical acellular subunit approach. Belisle has demonstrated the protective efficacy of a membrane fraction of Francisella combined with the adjuvant CLDC against Schu S4 challenge (61). A 17-kDa lipoprotein, a 43-kDa OMP, and heat shock protein 60 have been studied in murine models (62). These proteins were not protective in murine tularemia. Purified proteins, FopA and a 17-kDa lipoprotein, TUL4, were immunogenic but not protective (63,64). The fact that only some mouse strains are protected by vaccination with LVS suggests that it possesses a limited number of MHC-restricted protective antigens (8). To develop a licensable vaccine for tularemia, it may be necessary to give several proteins to elicit full response, rather than a poorly defined cocktail. The porin protein PorB from N. meningitidis, a TLR2 ligand, was able to enhance the protective response of Francisella tularensis LVS LPS from 25% to 70% of mice immunized, and later challenged intranasally with LVS four weeks after the last booster (65). In another study, the protective efficacy of isolated OMPs of Francisella tularensis, ethanol-inactivated LVS, or purified LVS LPS administered IP in Freund’s adjuvant were compared in a Schu S4 pulmonary challenge model. OMP immunization provided 50% survival at 20 days with a 1000fold decrease in bacterial loads in the liver and spleen (66). Conjugate Vaccines. To date, the only antigen identified as having a possible protective role in a subunit vaccine is LPS, which in Francisella lacks endotoxic activity (62). Immunization of mice with a vaccine comprised of Francisella tularensis LPS conjugated to bovine serum albumen protected against ID challenge with a strain of subspecies holarctica (type B), but had marginal protection against the same strain delivered as an aerosol, and had no protection with a strain of subspecies tularensis. (8) Since protection against virulent strains may require T-cell mediated immunity, investigators have considered conjugation to an antigen capable of eliciting T-cell immunity. A novel strategy pursued by investigators at Epivax is the use of mixtures of T-cell epitopes identified by genomics approaches delivered in a heterologous prime-boost regimen consisting of DNA vaccine prime and peptide boost (67). This vaccine was able to protect 50% of immunized mice from a lethal aerosol challenge with LVS. While earlier literature had described a capsule, such a structure had not been definitively described. Preliminary

studies with a capsule-like carbohydrate conjugated to a protein carrier are under current study.

Additional Strategies As is the case for many subunit vaccines, those for tularemia may need the addition of adjuvants. While DNA vaccines have been examined experimentally for some select agents such as anthrax, there are no reports for such genetic vaccines for tularemia. Ultimately, it may be necessary to assess primeboost strategies for tularemia vaccines. Such an approach may enable the priming of the immune system by delivery of a tularemia vaccine to a mucosal site, with a boost administered at the time of a suspected biological attack. Oral administration of LVS to mice induced both humoral and cellular responses as well as the induction of a short-lived protection against lethal systemic and respiratory infection with types A and B stains of F. tularensis compared to sham-immunized mice (68).

CONCLUDING REMARKS Many hurdles remain before an effective vaccine for tularemia could be licensed. Despite dramatic advances in genomics that have allowed the sequencing of both LVS and Schu S4 strains of F. tularensis, neither the basis for LVS attenuation nor the increased virulence of the Schu S4 strain have been determined. Recent advances in the ability to prepare defined mutations in the Schu S4 strain may now enable the development of a safe, protective type A-based vaccine. Alternatively, the identification of heretofore-undefined virulence factors may lead to the development of protective subunit vaccines. In either event, the difficulty in evaluating the efficacy of these vaccines in human clinical trials will place a burden on the identification of effective animal models in which to test these vaccines. Under current FDA guidelines, for a vaccine against a select agent to be approvable, it must demonstrate efficacy in at least two different animal species. This has posed a problem for vaccines against tularemia, since mice, unlike humans, are acutely susceptible to all subspecies of F. tularensis. To date, there have been few animal models considered adequate for testing of tularemia vaccines that may be predictive of success in humans.

REFERENCES 1. Francis E. Tularemia. In: Tice F, ed. Practice of Medicine. Vol 2. Hagerstown, MD: W. F. Prior Co., Inc. 1921:603–678. 2. Parker RR. Recent studies of tick-borne disease made at the United States Public Health Service Laboratory at Hamilton, Montana. Pac Sci Cong 1934; 3367–3374. 3. Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biological weapon: medical and public health management. JAMA 2001; 285:2763–2773. 4. Anonymous. 1970. Health aspects of chemical and biological weapons. World health Organization. Geneva, Switzerland. 5. Kaufmann AF, Meltzer MI, Schmid GP. The economic impact of a bioterrorist attack: are prevention and postattack intervention programs justifiable? Emerg Infect Dis 1997; 3:83–94. 6. Olsufiev NG, Emelyanova OS, Dunaeva TN. Comparative studies of strains of B. tularense in the old and new world and their taxonomy. J Hyg Epidemiol Microbiol Immunol (Prague) 1959; 3:138–149. 7. Saslaw S, Eigelsbach HT, Prior JA, et al. Tularemia vaccine study II. Respiratory challenge. Arch Intern Med 1961; 107:702–714. 8. Conlan JW, Oyston PCF. Vaccines against Francisella tularensis. Ann N Y Acad Sci 2007; 1105:325–350.

Chapter 78: Tularemia Vaccines 9. Mariathasan S, Weiss DS, Dixit VM, et al. Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis. J Exp Med 2006; 202(8):1043–1049. 10. Cole LE, Santiago A, Barry E, et al. Macrophage proinflammatory response to Francisella tularensis live vaccine strain requires coordination of multiple signaling pathways. J Immunol 2008; 180(10): 6885–6891. 11. Forslund A-L, Kuoppa K, Svensson K, et al. Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis. Mol Microbiol 2006; 69: 1818–1850. 12. Gil H, Platz GJ, Forestal CA, et al. Deletion of TolC orthologs in Francisella tularensis identifies role in multidrug resistance and virulence. Proc Natl Acad Sci U S A 2006; 103:12897–12902. 13. Clay CD, Soni S, Gunn JS, et al. Evasion of complement-mediated lysis and complement C3 deposition are regulated by Francisella tularensis lipopolysaccharide O antigen. J Immunol 2008; 181(8): 5568–5578. 14. Bosio CM, Bielefeldt-Ohmann H, Belisle JT. Active suppression of the pulmonary immune response by Francisella tularensis Schu4. J Immunol 2007; 178(7):4538–4547. 15. Elkins KE, Cowley SC, Bosio CM. Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain. Microbes Infect 2003; 5:135–142. 16. McCaffrey RL, Allen L-AH. Pivotal advance: Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape. J Leukoc Biol 2006; 80:1224–1230. 17. Malik M, Bakshi CS, McCabe K, et al. Matrix metalloproteinase 9 activity enhances host susceptibility to pulmonary infection with type A and B strains of Francisella tularensis. J Immunol 2007; 178:1013–1020. 18. Poquet Y, Kroca M, Halary F, et al. Expansion of Vgamma9Vdelta2 T cells is triggered by Francisella tularensis-derived phosphoantigens in tularemia but not after tularemia vaccination. Infect Immun 1998; 66:2107–2114. 19. Lavine CL, Clinton SR, Angelova-Fischer I, et al. Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity. Eur J Immunol 37(11):3007–3020. 20. Kirimanjeswara GS, Golden JM, Bakshi CS, et al. Prophylactic and therapeutic use of antibodies for protection against respiratory infecton with Francisella tularensis. J Immunol 2007; 179(1): 532–539. 21. Baron SD, Singh R, Metzger DW. Inactivated Francisella tularensis live vaccine strain protects against respiratory tularemia by intranasal vaccination in an immunoglobulin A-dependent fashion. Infect Immun 2007; 75(5):2152–2162. 22. Ericsson M, Sandstrom G, Sjostedt A, et al. Persistence of cell mediated immunity and decline of humoral immunity to the intracellular bacterium Francisella tularensis 25 years after natural infection. J Infect Dis 1994; 170:110–117. 23. Conlan JW, Shen H, Kuolee R, et al. Aerosol-, but not intradermalimmunizatoin with the live vaccine strain Francisella tularensis protects mice against subsequent aerosol challenge with a highly virulent Type A strain of the pathogen by an alphabeta T cell- and interferon gamma-dependent mechanism. Vaccine 2005; 23: 2477–2485. 24. Sjostedt A. Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations. Ann N Y Acad Sci 2007; 1105:1–29. 25. Kadull PJ, Reames HR, Coriell LL, et al. Studies on tularemia. V. Immunization of man. J Immunol 1950; 65:425–435. 26. Overholt EL, Tigertt WD, Kadull PJ, et al. An analysis of 42 cases of laboratory-acquired tularemia: treatment with broad spectrum antibiotics. Am J Med 1961; 30:785–806. 27. Burke DS. Immunization against tularemia. Analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J Infect Dis 1977; 135:55–60. 28. McCrumb FR, Snyder MJ, Woodward TE. Studies on human infection with Pasteurella tularensis. Comparison of streptomycin

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51. Pechous RD, McCarthy TR, Mohapatra NP, et al. A Francisella tularensis Schu S4 purine auxotroph is highly attenuated in mice but offers limited protection against homologous intranasal challenge. PLoS ONE 2008; 3:e2487. 52. Qin A, Scott DW, Mann BJ. Francisella tularensis tularensis Schu S4 Disulfide bond formation protein B, (DsbB), but not an RND-type efflux pump, is required for virulence. Infect Immun 2008; 76:3086. 53. Qin, A, Scott DW, Mann, BJ. Identificiation of an essential Francisella subsp. tularensis virulence factor. Infect Immun 2009; 77:152. 54. Twine S, Bystrom M, Chen W, et al. A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58kilodalton protein is attenuated for virulence and is an effective live vaccine. Infect Immun 2005; 73:8345–8352. 55. Thomas RM, Titball RW, Oyston PC, et al. The immunologically distinct O antigens from Francisella tularensis subspecies tularensis and Francisella novicida are both virulence determinants and protective antigens. Infect Immun 2007; 75:371–378. 56. Kay W, Petersen BO, Duus JO, et al. Characteristics of lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria. FEBS J 2006; 273:3002–3013. 57. Qin A, Mann BJ. Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2. BMC Microbiol 2006; 6:69. 58. Santiago A, Levine M, Barry E. Characterization of F. tularensis SchuS4 strain derivatives harboring deletions in essential intracellular genes. Abstracts of the 2008 Tularemia Workshop, March 30– April 2008, Bolton Landing, NY, p. 108. 59. Sjostedt A, Sandstrom G, Tarnvik A. Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium. Infect Immun 1992; 60:2855–2862.

60. Jia Q, Lee BY, Clemens DL, et al. Recombinant attenuated Listeria monocytogenes vaccine expressing Francisella tularensis IglC induces protection in mice against aerosolized Type A F. tularensis. Vaccine 2009; 27:1216–1229. 61. Belisle J. Generation of protective immunity against Francisella tularensis Schu4 using acellular vaccines. Abstracts of the 2008 Tularemia Workshop, March 30–April 2008, Bolton Landing, NY, p. 49. 62. Griffin KF, Oyston PCF, Titball RW. Minireview: Francisella tularensis vaccines. FEMS Immunol Med Microbiol 2007; 49:315–323. 63. Fulop M, Manchee R, Titball R. Role of lipopolysaccharide and a major outer membrane protein from Francisella tularensis in the induction of immunity against tularemia. Vaccine 1995; 13: 1220–1225. 64. Golovliov I, Ericsson M, Akerblom L, et al. Adjuvanticity of ISCOMs incorporating a T cell reactive lipoprotein of the facultative intracellualar pathogen, Francisella tularensis. Vaccine 1995; 13:261–267. 65. Chiavolini D, Weir S, Murphy JR, et al. Neisseria meningitidis PorB, a Toll-like receptor 2 ligand, improves the capacity of Francisella tularensis lipopolysaccharide to protect mice against experimental tularemia. Clin Vaccine Immunol 2008; 15(9):1322–1329. 66. Huntley JF, Conley PG, Rasko DA, et al. Native outer membrane proteins protect mice against pulmonary challenge with virulent type A Francisella tularensis. Infect Immun 2008; 76(8):3664–3671. 67. McMurry JA, Moise L, Gregory S, et al. High throughput validation of predicted T-cell epitopes in F. tularensis: the development of a novel T-cell epitope-based tularemia vaccine. Abstracts of the 2008 Tularemia Workshop, March 30-April 2008, Bolton Landing, NY, p. 50. 68. KuoLee R, Harris G, Conlan JW, et al. Oral immunization of mice with the live vaccine strain (LVS) of Francisella tularensis protects mice against respiratory challenge with virulent type A F. tularensis. Vaccine 2007; 25(19):3781–3791.

79 Vaccines Against Plague Scott A. Lloyd Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

YERSINIA PESTIS AND PLAGUE There are three Yersinia species pathogenic for humans: Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis. Y. enterocolitica and Y. pseudotuberculosis are enteropathogens found in contaminated food and water that cause self-limited gastroenteritis and mesenteric lymphadenitis (1,2). Y. pestis evolved from Y. pseudotuberculosis within the last 20,000 years (3) and is the causative agent of plague, a disease that has taken a devastating toll on human populations throughout history (4–6). In contrast to enteropathogenic yersiniae, Y. pestis is spread from rodents to humans by infected fleas. Following inoculation via a flea bite, bacteria migrate to the draining lymph node where they rapidly proliferate, thereby resulting in the formation of a bubo. Secondary septicemic plague occurs if bacteria subsequently disseminate into the bloodstream where their rapid proliferation will ultimately result in endotoxic shock. Secondary pneumonic plague, which comprises about 10% of cases, results when bacteria reach the lungs. In this instance, person-to-person transmission of Y. pestis can occur via infectious aerosols, thereby resulting in primary pneumonic plague. Primary pneumonic plague has a short incubation period (one to three days) and, in the absence of treatment, its mortality rate approaches 100% (4–6). While rare, outbreaks of pneumonic plague can be disastrous. The largest reported pneumonic plague epidemics occurred in Manchuria during 1910 to 1911 and 1920 to 1921 and resulted in an estimated 100,000 deaths (7,8). Because of its extreme virulence, Y. pestis has the distinction of being one of the few biological agents that has been deployed in warfare, as the Japanese military used infected fleas to trigger plague outbreaks in Chinese villages during World War II (9). Today, Y. pestis remains a prime candidate for use in a biowarfare or bioterrorist attack. This is, in part, because of the fact that researchers in the former Soviet Union successfully generated infectious aerosols of Y. pestis during the Cold War (10). More disturbingly, multiply antibiotic-resistant strains also were created (10), and naturally occurring antibiotic-resistant strains have also been found (11). The World Health Organization estimated that the release of 50 kg of aerosolized plague bacilli over a city of five million people would result in 150,000 cases of pneumonic plague and 36,000 fatalities (9). It is important to note that even a much smaller release of Y. pestis could cause widespread panic in a population as evidenced during recent pneumonic plague outbreaks in Surat, India, in 1994 (12) and in the Democratic Republic of Congo in 2005 (13–15). The high mortality rate,

the potential for person-to-person transmission, and the potential for its weaponization led the Centers for Disease Control to list Y. pestis as a category A biological warfare agent.

Y. PESTIS VIRULENCE FACTORS AND PROTECTIVE ANTIGENS Despite causing diseases that differ greatly in nature and severity, all three pathogenic Yersinia species share a 70 kilobase (kb) plasmid (called pCD1, pIB1, or pYV) that is required for virulence. The virulence plasmid encodes a type III secretion system (T3SS) as well as secreted effector proteins referred to as Yops (Yersinia outer proteins) (4,16–21). At 378C, and prior to eukaryotic cell contact, the Yersinia T3SS is expressed and forms a needle-like structure, comprised of YscF (Yop secretion protein F), on the bacterial cell surface (22). One other protein known to be surfacelocalized is LcrV (low calcium response protein V) (23), also known as the V (virulence) antigen (24), which sits at the tip of the T3SS needle structure (25). LcrV is required for the translocation of six Yop effectors into eukaryotic cells (23,26–29). Together, these effectors downregulate the host immune response, induce apoptosis, and disrupt the actin cytoskeleton of host cells, thereby inhibiting phagocytosis (16–21). An lcrV mutant is unable to inject these infectors proteins (23,26–28) and is, consequently, avirulent (30,31). Importantly, LcrV is a plague-protective antigen (6) and anti-LcrV antibodies that provide passive protection against plague (32–34) also block the translocation of Yop effectors (23,29,35) by promoting phagocytosis of the bacteria (29,35). It should also be noted that immunization with the needle component YscF has recently been shown to confer protection against plague (36,37), although the level of protection provided was much lower than that observed for LcrV. In addition to its role in the translocation of Yop effectors, several studies have also proposed that LcrV itself is an immunomodulatory protein that serves to suppress host inflammatory responses. Brubaker and coworkers showed that Y. pseudotuberculosis LcrV suppressed tumor necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-g) production by mice following challenge with avirulent LcrV-deficient Y. pestis (38). Subsequent studies using LcrV from Y. enterocolitica showed that LcrV triggering interleukin 10 (IL-10) production by interacting with TLR2 (Toll-like receptor 2) and CD14 and that TLR2
/
knockout mice are more resistant to Yersinia infection (39,40). Similarly, Overheim et al. demonstrated that Y. pestis LcrV induces IL-10 production and suppresses lipopolysaccharide (LPS)-induced TNF-a production in macrophages (41). However, recent studies

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have demonstrated that LcrV does not possess any immunomodulatory activity and that TLR2 and IL-10 do not play significant roles in the host immune response against Yersinia. Specifically, Philipovskiy et al. showed that anti-LcrV antibodies inhibited the growth of Y. pestis in IL-10–/–- knockout mice and that the protective effect of the anti-LcrV antibody was solely due to its ability to abrogate Yop translocation (42). In addition, Auerbuch and Isberg demonstrated that IL-10 is undetectable in mouse tissues following Y. pseudotuberculosis infection until the late stages of infection. This increase in IL-10 occurred independently of TLR2 and LcrV and was in fact ameliorated by the type III effector YopJ (43). Other studies using Y. pestis have also shown very little, if any, induction of IL-10 in mouse tissues following infection (44–46). Furthermore, Goguen and coworkers demonstrated that the ability of Y. pestis LcrV to stimulate TLR2 in vitro was due to a minor component of the recombinant LcrV preparation that consists of high molecular weight multimers and aggregates. Because of this fact, they could not rule out that contaminants in the LcrV multimer fraction were responsible for the TLR2 stimulating activity. Finally, these authors demonstrated that there was no discernible difference between wild-type and TLR2–/– knockout mice in severity of disease or kinetics of survival following subcutaneous challenge with Y. pestis (47). On the basis of these studies, one must conclude that LcrV is not an immunomodulatory protein and that its primary function is in the translocation of Yop effectors. In addition to the 70 kb virulence plasmid, Y. pestis contains two other virulence-associated plasmids (48). The first is the 9 kb pPst (or pPCP1) plasmid that encodes the Pla plasminogen activator protein (49), a surface protease that is required for bacterial dissemination in both bubonic (50,51) and pneumonic forms of the disease (52). However, Pla is not a protective antigen as mice immunized with Pla in the form of a DNA vaccine were not protected against plague (53). The other plasmid is the 100 kb pMT1 (or pFra) plasmid, which encodes the murine toxin required for survival in the flea (54), and also the fraction I (F1) capsular antigen (55), which forms surface a capsule active in inhibiting phagocytosis (56,57). The F1 antigen is a protective antigen against plague (6) and antibodies against F1 may promote opsonin-mediated phagocytosis of the bacteria (57). The F1 antigen, however, is dispensable for virulence as F1-negative strains are almost fully virulent in mice, nonhuman primates, and humans, whether delivered subcutaneously or by aerosol (58–63).

PLAGUE PATHOLOGY Rodents, nonhuman primates, and humans exhibit similar pathologies in response to infection with Y. pestis. Using a Brown Norway rat model of bubonic plague, Sebbane et al. extensively studied the kinetics of disease progression and the host immune response following intradermal inoculation of Y. pestis in the left lower back of the animals (46). In this model, the left inguinal (proximal) lymph node was colonized as early as six hours post infection in some rats and in all rats by 24 to 36 hours. After 36 to 72 hours, bacteria appeared in the left axillary (distal) lymph node and the spleen. A detailed analysis of the proximal draining lymph node revealed that a few extracellular bacteria appeared in the marginal sinus within 24 hours. Bacteria continued to increase in numbers and continued to spread within the marginal sinus, however there was only a limited recruitment of polymorphonuclear leukocytes (PMNs) at this time. By 36 hours, multifocal aggregates of bacteria

extended into the cortex and were surrounded by increasing numbers of PMNs. By 72 hours, the normal architecture of the lymph node was replaced by large numbers of bacteria, cellular debris, necrotic PMNs, and fibrin. Evidence of fibrin thrombi and hemorrhage were also present. Ultimately, bacteria colonized the entire node, resulting in hemorrhage, septicemia, and necrotizing vasculitis. Typical abscesses did not develop even though PMNs co-localized with bacterial aggregates. Small numbers of macrophages were recruited to the primary lymph node within 18 hours, but their numbers decreased thereafter. This was presumably due to apoptosis as an increase in caspase-positive cells was observed in the bubo at later time points; a subsequent study demonstrated that the type III effector YopJ contributes to apoptosis in the bubo (64). In addition, elevated levels of IFN-g and TNF-a were detected only after bacteremia had developed. Notably, elevated IL-10 levels were only detected in a single rat. Detailed analysis of a murine model of pneumonic plague has also been performed (45). Using intranasal administration of Y. pestis cultivated at 378C, they showed that the infection began with an anti-inflammatory state that lasts 24 to 36 hours. By 48 hours, the infection proceeded to a highly inflammatory state and by 72 hours the mice began to die from a purulent multifocal exudative bronchopneumonia that closely resembled pneumonic plague in humans. One hour following inoculation of 104 bacteria, 103 bacteria could be recovered from the lungs of mice, which is consistent with a previous study of pneumonic plague in monkeys (65). Bacterial numbers in the lung proceeded to increase steadily at a rapid rate, reaching 1010 by day 3. Within 24 to 36 hours, bacteria disseminated to the spleen and other tissues, where they proliferated to high numbers by 72 hours. At 24 hours post infection, the lungs did not exhibit any signs of pathology. In addition, only low levels of TNF-a and IFN-g were observed at this time point. These low cytokine levels are likely due to the effects of the T3SS as Nakajima and Brubaker observed high levels of these cytokines in mice infected with a Y. pestis strain lacking the pCD1 virulence plasmid that encodes the T3SS, but not with a wild-type strain (66). Importantly, elevated IL-10 levels were only observed in a single mouse. By 48 hours, exudates were present in the smaller bronchi, and the bronchi appeared hyperplastic. Extracellular bacteria were present in the alveoli and small bronchioles at this time. In addition, foci of neutrophils were observed surrounding the smaller bronchi and a decrease in the numbers of alveolar macrophages was seen. By 72 hours, extensive hemorrhages with large numbers of neutrophils were also evident and large areas of the lungs were filled with masses of extracellular bacteria.

THE YERSINIA LIFESTYLE On the basis of the preceding data, one can easily conclude that Y. pestis uses the F1 capsular antigen and its T3SS, of which LcrV is a part, to avoid phagocytosis and suppress cytokine production by cells responsible for generating the innate immune response, thereby allowing the bacteria to live the extracellular lifestyle. Indeed, the Y. pestis T3SS has been shown to target dendritic cells, macrophages, and neutrophils in vivo (67). In addition, early studies by Burrows and Bacon (68) demonstrated that a virulent Y. pestis strain grown at 288C, at which temperature F1, LcrV, and the rest of the T3SS are not expressed, are phagocytosed and destroyed by PMNs. Furthermore, antibodies against F1 and LcrV are known to enable

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phagocytosis of the bacteria (29,35,56,57), leading one to conclude that the humoral immune response is critical for protection against plague. Although this is undoubtedly true, other studies paint a more complex picture of plague pathogenesis and suggest that the ability of cellular immune responses to clear intracellularly replicating bacteria is also an important factor. For example, work by Cavanaugh and Randall (56) confirmed and extended prior findings by demonstrating that plague bacilli expressing F1 and LcrV are indeed resistant to phagocytosis by PMNs and monocytes. In addition, bacteria only expressing LcrV were resistant to phagocytosis by PMNs but were readily ingested by monocytes, while bacteria expressing neither F1 nor LcrV were phagocytosed by both cell types. However, they showed that while PMNs killed ingested bacteria, monocytes failed to do so and, in fact, bacteria within monocytes were able to replicate extensively. Following growth in monocytes, the released bacteria expressed both F1 and LcrV. Together, these results suggest that monocytes may constitute an initial protective niche for Y. pestis, thereby allowing the bacteria time to express F1 and LcrV. Consistent with this view, Lukaszewski et al. (69) demonstrated that, following subcutaneous delivery of Y. pestis grown at 288C, bacteria were able to survive and replicate within splenic macrophages until the later stages of infection, at which time extracellular bacteria were observed. Likewise, Finegold (65) reported finding intracellular bacteria even in late stages of infection following aerosol exposure of rhesus monkeys using bacteria cultured at 288C. However, Lukazewski et al. (69) demonstrated that pretreatment of macrophages with TNF-a and IFN-g restricted intracellular replication. In addition, a prior study by Nakajima and Brubaker (66) showed that mice dosed with TNF-a and IFN-g were protected against intravenous challenge with a non-pigmented (pgm-) strain of Y. pestis. The aforementioned studies suggest that Th1 type cytokine responses are an important component in the immune response against plague. These findings were extended by Smiley and coworkers who demonstrated that vaccination of mice with an attenuated (pgm-) strain of Y. pestis primes T cells that provide passive protection against intranasal challenge with the same strain (70). Similarly, vaccination of B cell– deficient mMT mice with an attenuated Y. pestis strain also conferred protection against challenge (71). While clearly demonstrating a role for T cells in the immune response against plague, the results should be interpreted with some caution as Pujol et al. (72) demonstrated that the ripA gene, which is encoded within the pgm locus, suppresses nitric oxide production by IFN-g-activated macrophages and is required for intracellular replication. This suggests that pigmentation-negative Y. pestis strains may therefore be more sensitive to cell-mediated immune responses than wild-type strains. In addition, it should be pointed out that passive transfer of T cells has not been shown to confer protection against a wild-type strain when administered via the aerosol route. Nevertheless, these studies suggest that while antibodies against F1 and LcrV promote phagocytosis, cytokine production by T cells promotes the killing of bacteria that have been ingested by macrophages.

KILLED WHOLE-CELL VACCINES Despite the inherent dangers of Y. pestis, no suitable vaccine exists (6). A killed whole-cell vaccine was first developed by Haffkine in 1897. Although the vaccine was reported to be effective against bubonic plague, it was highly reactogenic (73).

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Symptoms included pain, swelling, erythema, and regional lympadenopathy. For these reasons, such vaccines largely fell out of favor until Meyer and colleagues developed a less reactogenic killed whole-cell vaccine (eventually called Plague Vaccine, USP), which served as a vaccine for the U.S. military since the 1940s. Military personnel in WWII and Vietnam who were vaccinated did not contract plague (74). Despite these results, Plague Vaccine, USP still retained unwanted reactogenicity, required multiple boosts, and is no longer currently manufactured (6). In addition to their unwanted side effects, the chief limitation of killed whole-cell vaccines is their inability to protect against pneumonic plague in animals (75) and humans (58).

LIVE-ATTENUATED VACCINES The first, and so far only, live bacterial plague vaccines to be used in humans are the attenuated Y. pestis vaccine strains EV (and its derivatives) and Tjiwidej (74). These strains were widely used in plague pandemic regions of Africa and Asia and vaccination programs using these strains were shown to reduce the incidence of plague in these areas. The EV76 strain, which lacks the 102 kb chromosomal pigmentation (pgm) locus, confers protection against both bubonic and pneumonic plague (76,77). However, this strain, while essentially avirulent by the subcutaneous route in rodents, is lethal to nonhuman primates at moderate doses and causes significant side effects in humans (76). More recently, a Y. pestis strain that lacks both the Pla protease and the pgm locus, and which is less virulent than the EV76 strain, was shown to induce a humoral immune response against F1 in African green monkeys following aerosol delivery (78). Whether this immune response is protective is not yet known. Other strategies to generate live plague vaccines have involved the introduction of a lpxM mutation into the EV strain background, thereby generating a strain that synthesizes a less toxic penta-acylated LPS (79), the creation of a DNA adenine methylase dam mutant (80), and deletion of the type III effector YopH (81) from wild-type Y. pestis strains. While these strains were immunogenic and conferred protection against subsequent challenge with a wild-type strain, the development of live attenuated Y. pestis strains that are safe and immunogenic in humans faces a high hurdle.

SUBUNIT VACCINES It has long been established that immunization with F1 purified from Y. pestis protects animals against bubonic (82) and pneumonic (83) plague. More recently, purified recombinant F1 was shown to confer protection against pneumonic plague in mice (84). However, the F1 antigen is dispensable for virulence (58,59,61–63). Therefore, vaccines based solely upon F1 will not confer protection against almost fully virulent F1-negative strains. For this reason, researchers have also developed LcrVbased subunit vaccines, as LcrV is an indispensable virulence determinant (30,31) and has long been known to be a protective antigen (85). Many studies demonstrated that passive immunization with antibodies directed against LcrV confers protection against plague (32–34,86). Similarly, protection is observed following immunization with purified recombinant LcrV (87,88). Although LcrV is an indispensable virulence determinant, sequence analyses of LcrV from Y. enterocolitica, Y. pseudotuberculosis, and Y. pestis revealed a region of LcrV whose sequence is variable, particularly in O:8 serotype strains

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of Y. enterocolitica. Antibodies generated against the O:8 LcrV do not provide substantial passive protection against strains expressing other LcrV variants (89). For this reason, researchers have focused on generating subunit vaccines that consist of both F1 and LcrV, which should be more difficult to circumvent. In fact, such vaccines provide enhanced protection against plague (90). Importantly, immunization with an admixed preparation of F1 and LcrV protects against aerosol challenge (75,91). The combined IgG1 titer to F1 and LcrV correlates with protection against plague in mice (92). Similarly, immunization with an F1-LcrV fusion protein protects mice against challenge with F1-positive and F1-negative strains (93,94). The F1-LcrV fusion protein also conferred high levels of protection in cynomolgus macaques against aerosolized Y. pestis. However, only low levels of protection were observed in African green monkeys, possibly because of more variable immune responses against LcrV (95). Antibody titers to the F1-LcrV fusion protein did not correlate with protection in African green monkeys. For this reason, researchers developed an in vitro system to identify correlates of protection. These models take advantage of the fact that pathogenic yersinae induce a cytotoxic effect in macrophages, as evidenced by changes in cellular morphology, the production of the apoptosis-specific enzyme caspase-3 and release of the cytosolic enzyme lactic acid dehyrdrogenase. Antibodies against LcrV neutralize these cytotoxic effects by blocking the injection of type III effectors (23,29,35), and the neutralizing activity of antiLcrV antibodies correlates with protection in both mice (96,97) and nonhuman primates (98). Recently, the combined F1 þ LcrV subunit vaccine was tested in a phase I clinical trial (99). Groups of individuals were immunized on days 1 and 21, with either 5, 10, 20, or 40 mg of F1 and LcrV (admixed in alum in a 2:1 molar ratio). Antibodies to both F1 and LcrV were produced within two weeks of the first dose and increased after the booster dose on day 21, although there was a large variation in antibody responses between individuals within each dose group. Passive transfer of protective immunity correlated with total combined IgG titers to F1 and LcrV on day 21. Importantly, this subunit vaccine was not associated with adverse side effects. However, IgG titers declined by day 70 in all dose groups.

NEW APPROACHES Given the relatively low and variable immune responses observed with the F1 þ LcrV subunit vaccine, much attention has been focused recently on developing new methods to generate more robust immune responses against these antigens. These include the use of DNA vaccines (53,99–101), the use of viral vectors for antigen delivery (100,102), encapsulation in microspheres (103,104), the use of adjuvants other than alum (105–107), different routes of administration (103,107), heterologous prime-boost vaccination regimens (108), expression of F1 and LcrV in attenuated Salmonella vaccine vectors (109–112), and use of attenuated strains of the closely related enteric pathogen Y. pseudotuberculosis (113–115). While potentially promising, many of these approaches have serious limitations. For instance, DNA vaccines often require multiple immunizations (sometimes using ‘‘Gene Gun’’ technology) and have yet to show promising results in humans. Viral vectors and other forms of intranasal delivery have possible safety concerns. Testing new adjuvants will require substantial further research. Many of the aforementioned Salmonella vaccine vectors have yet to be tested in humans. Finally, attenuated Y. pseudotuberculosis

strains run the risk of causing post-infectious sequelae such as reactive arthritis (116), particularly in HLA-B27positive individuals (117).

CHARACTERISTICS OF AN IDEAL PLAGUE VACCINE Clearly, there remains an immediate need to develop new and improved plague vaccines. One can envision several characteristics of the ideal plague vaccine regimen: A strong broad protective immune response—The ideal vaccine regimen must induce a very high level of protection. Biological attack could subject at least some victims to levels of exposure far higher than those seen after natural infection. Attacks are likely to be via the respiratory route, and thus some component of mucosal immunity may be helpful; in a pneumonic plague outbreak, some victims succumbed with mucosal and not pulmonary pathology (8). Cell-mediated immunity to the intracellular Y. pestis should also be beneficial; mice deficient in antibody responses can be protected by vaccination (71). Vaccines should engender a rapid response and the potential to augment the response still further by administering a booster dose. Ease of rapid administration—Implementation of a plague vaccine regimen will likely require either vaccination of large numbers of individuals or, in the event of release, very rapid vaccination of a small but dense population. In either case, ease of administration (including a convenient supply chain) would be a great benefit. The optimal vaccine should be deliverable by personnel with limited formal expertise. Rapid and cost-effective production—Since the government will likely need to produce and maintain large stocks of vaccines against several agents, each vaccine should be inexpensive to produce and should have a reasonably long shelf life. Multivalency, protection against more than one infection in a single vaccine, would be advantageous. Rapid production would also be helpful, so that the vaccine can be made quickly in response to a manifest threat. Perhaps an even greater advantage would be if the vaccine provided some positive externality, such as protection against another infection whose prevention appealed to an established and predictable market. High degree of safety—It is likely that in the event of release, or prior thereto, we will need to vaccinate large numbers of people who will probably never actually encounter the agent. Therefore, it will be important to assure that the vaccine does not cause a greater degree of cumulative morbidity than the agent it is designed to thwart.

THE PROMISE OF PRIME-BOOST VACCINATION As described above, subunit vaccines are in advanced stages of development. However, we feel that the subunit vaccines currently under evaluation will not provide optimal protection against these agents. In addition, they will not engender mucosal or cellular immunity, nor are they convenient to administer rapidly or in multiple-dose regimens. Live-attenuated mucosal vaccines have all of the advantages sought in the ideal biodefense vaccine, except that the magnitude of the immune response has not yet reached the level required for biodefense. Thus, there exists the possibility of combining two of the above

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strategies to exploit their respective strengths and circumvent their weaknesses. Indeed, mucosal prime-parenteral boost strategies so far attempted have suggested that they can provide high levels of protection, a broad immune response including Th1, Th2 and mucosal immunity, and reasonable convenience. The promise of enteral prime/parenteral boost was first supported by human studies using oral polio vaccine (OPV) and parenteral inactivated polio vaccine (IPV) recipients boosted with either OPV or IPV (118). In this study, parenteral IPV vaccination was able to boost systemic and mucosal IgA responses in previously OPV-vaccinated subjects, but not IPV vaccinated subjects. Interestingly, IPV vaccination of OPV ‘‘primed’’ subjects, though immunized decades earlier, mounted rapid (by day 7) and vigorous IgG as well as IgA responses in the periphery as well as at the mucosal level. In other words, IPV boosting of OPV primed individuals generated broader immune responses that were at least as vigorous in all aspects as those both primed and boosted with the parenteral vaccine. These human data have subsequently been reproduced in mouse models with very different mucosal vaccines (119–121). Several observations can be made from this literature. Jespersgaard (119) and Londono et al. (120) demonstrated that mucosal prime/parenteral boost resulted in boosting of systemic responses with good Th1/Th2 balance and boosting of secretory IgA. At the same time, Anderson et al. (122) and Foynes et al. (123) demonstrated that effective boosting can take place in the presence of only very weak responses to the initial prime (i.e., the mucosa may be primed before it mounts a demonstrable antibody response). Using Salmonella Typhimurium expressing the C-terminal domain of Clostridium difficile toxin A (cytoplasmically), Ward et al. (121) showed that mice primed per os with the Salmonella vaccine mounted vigorous systemic antibody responses to exquisitely small subcutaneous doses of the purified protein, doses so small as to generate no antibody responses when given alone. Taken together, the data support the utility of enteral prime/parenteral boost. Such prime/boost strategies are likely to provide rapid, vigorous, and very broad immune responses, including cell-mediated, antibody-mediated and mucosal components. In addition, it will be possible to prime a large population by a safe and easily administered oral route, then boost them parenterally at a later time, thereby permitting them to mount significantly stronger, faster, and more balanced responses than they would in the absence of such enteral priming. This strategy could enable a rapid and effective public health response strategy.

CONCLUSIONS Plague vaccine development made slow progress for many years, but recent interest in biodefense has provided a major impetus to this research. It is anticipated that at least one new plague vaccine candidate will enter advanced clinical trials within the next several years, ultimately leading to the development of a safe and effective vaccination strategy.

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Chapter 79: Vaccines Against Plague 75. Williamson ED, Eley SM, Stagg AJ, et al. A sub-unit vaccine elicits IgG in serum, spleen cell cultures and bronchial washings and protects immunized animals against pneumonic plague. Vaccine 1997; 15:1079–1084. 76. Meyer KF, Smith G, Foster L, et al. Live, attenuated Yersinia pestis vaccine: virulent in nonhuman primates, harmless to guinea pigs. J Infect Dis 1974; 129(suppl):S85–S112. 77. Russell P, Eley SM, Hibbs SE, et al. A comparison of Plague vaccine, USP and EV76 vaccine induced protection against Yersinia pestis in a murine model. Vaccine 1995; 13:1551–1556. 78. Welkos S, Pitt ML, Martinez M, et al. Determination of the virulence of the pigmentation-deficient and pigmentation-/plasminogen activator-deficient strains of Yersinia pestis in non-human primate and mouse models of pneumonic plague. Vaccine 2002; 20:2206–2214. 79. Feodorova VA, Pan’kina LN, Savostina EP, et al. A Yersinia pestis lpxM-mutant live vaccine induces enhanced immunity against bubonic plague in mice and guinea pigs. Vaccine 2007; 25:7620–7628. 80. Robinson VL, Oyston PC, Titball RW. A dam mutant of Yersinia pestis is attenuated and induces protection against plague. FEMS Microbiol Lett 2005; 252:251–256. 81. Bubeck SS, Dube PH. Yersinia pestis CO92 delta yopH is a potent live, attenuated plague vaccine. Clin Vaccine Immunol 2007; 14:1235–1238. 82. Baker EE, Sommer H, Foster LE, et al. Studies on immunization against plague. I. The isolation and characterization of the soluble antigen of Pasteurella pestis. J Immunol 1952; 68:131–145. 83. Ehrenkranz NJ, Meyer KF. Studies on immunization against plague. VIII. Study of three immunizing preparations in protecting primates against pneumonic plague. J Infect Dis 1955; 96:138–144. 84. Andrews GP, Heath DG, Anderson GW Jr., et al. Fraction 1 capsular antigen (F1) purification from Yersinia pestis CO92 and from an Escherichia coli recombinant strain and efficacy against lethal plague challenge. Infect Immun 1996; 64:2180–2187. 85. Burrows TW, Bacon GA. The effects of loss of different virulence determinants on the virulence and immunogenicity of strains of Pasteurella pestis. Br J Exp Pathol 1958; 39:278–291. 86. Une T, Brubaker RR. Roles of V antigen in promoting virulence and immunity in yersiniae. J Immunol 1984; 133:2226–2230. 87. Anderson GW Jr., Leary SE, Williamson ED, et al. Recombinant V antigen protects mice against pneumonic and bubonic plague caused by F1-capsule-positive and -negative strains of Yersinia pestis. Infect Immun 1996; 64:4580–4585. 88. Leary SE, Williamson ED, Griffin KF, et al. Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague. Infect Immun 1995; 63:2854–2858. 89. Roggenkamp A, Geiger AM, Leitritz L, et al. Passive immunity to infection with Yersinia spp. mediated by anti-recombinant V antigen is dependent on polymorphism of V antigen. Infect Immun 1997; 65:446–451. 90. Williamson ED, Eley SM, Griffin KF, et al. A new improved subunit vaccine for plague: the basis of protection. FEMS Immunol Med Microbiol 1995; 12:223–230. 91. Williamson ED, Eley SM, Stagg AJ, et al. A single dose sub-unit vaccine protects against pneumonic plague. Vaccine 2000; 19:566–571. 92. Williamson ED, Vesey PM, Gillhespy KJ, et al. An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model. Clin Exp Immunol 1999; 116:107–114. 93. Anderson GW Jr., Heath DG, Bolt CR, et al. Short- and long-term efficacy of single-dose subunit vaccines against Yersinia pestis in mice. Am J Trop Med Hyg 1998; 58:793–799. 94. Heath DG, Anderson GW Jr., Mauro JM, et al. Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine. Vaccine 1998; 16:1131–1137. 95. Pitt LM. Non-human primates as a model for pneumonic plague. FDA workshop on Animal Models and Correlates of Protection

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80 Development of Vaccines for Ebola and Marburg Viruses: Efficacy and Regulatory Considerations Gary J. Nabel Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

FILOVIRUS DISEASE: SYMPTOMS, PATHOGENESIS, AND CLINICAL COURSE The initial recognition of filoviruses dates back to 1967, when the first cases of Marburg virus were detected in an animal colony in Marburg, Germany (1). Identification of an outbreak of hemorrhagic fever in the Ebola River valley in 1976 led to the recognition of the Ebola virus, a closely related pathogen, both in terms of the disease and its genetic organization. After its discovery and a few sporadic incidents of Ebola fever in the 1980s, outbreaks have been seen more consistently since the mid-1990s, raising the concern of a new mode of transmission and an increasing public health threat (2,3). Disease symptoms of hemorrhagic fever associated with Ebola and Marburg viruses progress through four phases; initial symptoms of infection are fever, cough, and myalgia followed by the appearance of a characteristic rash and petechiae, and clotting abnormalities indicating liver dysfunction. Soon thereafter, signs of uncontrolled bleeding are apparent, particularly in the gastrointestinal tract. The disease progresses rapidly, reaching terminal phases within one to two weeks of infection, with hepatitis, aberrant clotting times, and diffuse bleeding, ultimately leading to hypovolemic shock without concurrent bacterial infection. The mortality rates for Ebola virus and the more recent strains of Marburg virus have generally ranged from 50% to 90%. The pathogenesis of the disease is likely related to the targeting of the virus to reticuloendothelial cells of the liver, lung, and spleen, and later dissemination to the endothelial cells. It has been postulated that direct cytopathic effects of the virus in cells of the monocytic lineage lead to release of cytokines from dying cells that lead to fever. It is the subsequent liver damage and compromise of the vascular endothelium accompanying the uncontrolled bleeding and loss of vasomotor tone that ultimately leads to septic shock (Table 1).

PAST OUTBREAKS AND EPIDEMIOLOGY Past outbreaks of Ebola virus infection have occurred in equatorial Africa, specifically Uganda, Sudan, Cote d’Ivoire, Republic of Congo, and the Democratic Republic of Congo (4). In equatorial Africa, the largest percentage of cases in recent years has appeared in the rainforest regions of the Congo and Uganda. A recent outbreak in Uganda during the fall of 2008 has led to the definition of a fifth Ebola virus strain, the Bundibugyo species, which is distinct from those previously

defined (5). Among the previous Ebola isolates, this strain is distant but most closely related to the Cote d’Ivoire virus: its divergence from previously known species posed a challenge in identifying the cause of the 2008 Uganda outbreak and demonstrated its continued evolution. This concern was raised again when a virus resembling the Reston strain was identified in pigs with hemorrhagic fever in the Philippines (6), documenting an additional animal that could serve as a reservoir of infection or as an intermediate host. This finding has also heightened concerns about the possibility that Ebola virus might affect the human food supply chain beyond the contact with bush meat in central Africa. Outbreaks of Marburg virus have been reported in Germany, Yugoslavia, South Africa, Kenya, the Soviet Union, and Democratic Republic of Congo. Recently, outbreaks of Marburg virus have been detected in Uganda and Angola (7). There has been a recent report of detection of virus in bat caves where victims were exposed (8), strongly suggesting that the bat serves as intermediate host for this virus. In contrast, the epidemiology of Ebola virus may have undergone a shift in the last decade or so. There is evidence of zoonotic transmission with multiple points of contact between humans and certain animals. Fruit bats have been previously implicated as the reservoir of infection (9), but its correlation with human outbreaks has not been established. Other data has suggested the likelihood more recently that virus is transmitted from nonhuman primates (NHPs) to humans, presumably because of bush meat hunters who become exposed to blood products from the great apes, and subsequently may transmit virus to family and village members (2,3). In addition, laboratory workers and medical personnel are at increased risk for infection, mostly from accidental needle sticks. These outbreaks of hemorrhagic fever, together with the recent swine infections in the Philippines, indicate that there is ongoing evolution and zoonotic transmission of these viruses, either from bats in the case of Marburg virus and bats, NHPs, and possibly swine for Ebola virus. Whether the swine represent an incidental host or a vector for this disease at the present time remains unknown.

VIROLOGY AND GENOME

The Ebola virus is composed of a genome that is *19 kb in size, and is a prototypic negative-strand RNA virus (10). The filovirus species is most closely related to the paramyxoviruses but

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Table 1 Features of Ebola and Marburg Viruses and Relevance to Vaccine Development

Five species:

First appearance: Mortality *CDC pathogen category:

Ebola

Marburg

Zaire Multiple Species: Sudan Cote D’Ivoire Reston Bundibugyo 1976 50–90% A

Popp Raven Angola Musoke Others 1967 30–90% A

*Centers for Disease Control.

have alternative genetic organization as well as morphology and mechanisms of replication. The virus encodes seven gene products, including the glycoprotein (GP) that is subject to significant posttranslational modification by both N- and O-glycosylation. Its molecular biology has been reviewed elsewhere in detail (10). The GP undergoes proteolytic cleavage that gives rise to an N-terminal GP1 and COOH-terminal GP2 region (11). The GP1 domain is thought to interact with a yetunidentified cellular receptor on monocytic and endothelial cells, while GP2 is involved in viral fusion that occurs in a pH-dependent fashion within endosomes. GP is present on the external surface of the viral particle and is responsible for binding to specific cell types of endothelial, hepatic, and monocytic origin (12,13). Though it appears that there is at least one well-defined attachment factor, DC-SIGN (14–16), the specific receptor for Ebola GP has not been defined. While a transferrin receptor has been implicated in facilitating viral entry (17), it is not thought to be the primary receptor, and the identification of such a protein remains a goal of current research. Additional insight into the mechanism of Ebola virus entry has come from the recognition that GP may be subject to endosomal proteolysis by cathepsin-related proteases after host cell binding (18), suggesting a multistep process required for cell fusion and additional possible targets of antiviral drugs or antibodies. Recently, an atomic-level structure of the Ebola virus GP complexed to a human neutralizing antibody has been determined (19). This information will facilitate efforts to understand the mode of entry by this virus and its potential utility as an immunogen for cellular and humoral immunity. In addition to the transmembrane form of the GP, which is a product of RNA editing, the normal open reading frame of the GP gene gives rise to a secreted form of the protein whose functions are not completely understood. While there have been several reports that suggest it may have an anti-inflammatory effect (11,12), it is found in high concentrations in the blood following infection, and its role in the pathophysiology of infection is not yet fully understood. In addition to the glycoprotein, the other major gene products of the virus include the L protein, which is the virus-specific polymerase as required for replication from the viral genomic RNA. The VP35 protein appears to serve as an antagonist of interferon-a and may function as a factor that increases its virulence in experimental animals (20). In addition, the nucleoprotein has an observed molecular mass of approximately 115 Kd, and appears to be a partially disordered protein whose biochemistry facilitates the formation of the filamentous structure of the virus (21). In addition, a combination of three viral proteins, including NP, VP35, and VP24, are required for

generation of viral-like particles (22). The VP40 protein serves both as a matrix protein that contributes to formation of the virus related to its essential role in budding (21,23). While there is approximately 55% amino acid similarity between Ebola viruses and Marburg virus, the Marburg virus genome encodes the same gene products and has a conserved overall structure and gene organization. However, unlike Ebola virus, it does not appear to give rise to a secreted GP, which may in part contribute to the difference in pathogenicity of these viruses in vivo.

PATHOGENESIS AND MECHANISMS OF PROTECTION IN ANIMAL MODELS While the initial cases of Ebola virus infection were observed in humans and subsequently in NHPs, the virus has been adapted to grow in several rodent models that have been useful in understanding some aspects of the pathophysiology of the disease. Although the virus causes disease in mice and guinea pigs, its lethality is diminished, and it is easier to protect against infection by vaccines in these models, probably because of the adaptation of the virus to alternative host receptors and cellular genes that may affect the replication potential of these viruses in rodents. A variety of studies have been performed in guinea pigs, which are vulnerable to infection. Although infection by primary isolates is not highly lethal, additional serial passages of Ebola and Marburg viruses have rendered these viruses more pathogenic (1,24–26), and they have been useful for the initial vaccine studies that demonstrated protective efficacy of gene-based vaccines (27). Data from the guinea pig model allowed definition of immune correlates using a rodent model with greater lethality, similar to the disease in NHPs and humans. Gene-based vaccination with GP and NP conferred protection in these animals (27). Immunization with these vectors induced potent CD8 T-cell responses and, in combination with the NP vector, elicited GP-specific binding antibodies by ELISA that provided a correlate of immune protection. It is important to note, however, that passive transfer of this hyperimmune sera from immunized animals did not confer protection to naı¨ve recipients (27). Since then, this correlate of vaccine protection has been observed in rodent challenges with GP and NP vaccines encoded by Venezuelan equine encephalitis (VEE) vectors (28,29). The ability to grow primary filoviruses in mice has proven more challenging. However, it has been possible to adapt the Zaire strain of Ebola virus to inbred mice, where it has proven to be highly lethal (30). These studies have led to a number of mechanistic studies regarding the role of different arms of the cellular immune system to contain or prevent infection in the murine model, indicating that there is a role for both cellular and humoral immunity for containment in mice (31–33). In addition, passive transfer studies using neutralizing monoclonal antibodies have shown an ability to protect in a murine model (34) but have failed to show protection in the NHP (35), indicating disparity between the different animal models with regard to the mechanism of protection. In NHPs, mortality after lethal challenge exceeds 90%, and the manifestations and time course of the disease, although varied among the different NHPs, are more reflective of the time course seen in humans (24,36–39). In the NHP, initial success showing protection against lethal challenge was demonstrated using gene-based vaccination by priming with

Chapter 80: Development of Vaccines for Ebola and Marburg Viruses

plasmid DNAs encoding GP and NP followed by boosting with replication-defective adenoviral (rAd) vectors. This method of immunization also elicited high titers of ELISA binding antibodies that served as a reliable correlate of protection in the NHP (40). Since then, it has been possible to use a single adenoviral vector to achieve this protection with a similar correlate of immune protection (41). Now, it is possible to achieve protection using other methods of delivery, including vesicular stomatitis virus (42), parainfluenza virus (43) or viruslike particles (44), and protection in these instances also correlates with the generation of GP-specific antibodies in vaccinated animals that survived lethal challenge. In addition, the vesicular stomatitis virus vectors have been shown to confer protection in animals when administered shortly after inoculation of the virus, raising the possibility that, like high-dose single-shot recombinant Ad vector, this vector may be useful in an acute outbreak setting. The role of T cells in mediating protection against filovirus infection is likely significant on the basis of the murine model of Ebola virus challenge. In this system, it is clear that CD8þ T cells are elicited by gene-based vaccination against NP (29,32,45), as well as against GP (27). Adoptive transfer of cytotoxic T lymphocytes (CTL) in naı¨ve mice can protect against lethal Ebola virus challenge (29). Similarly, with Marburg virus, protection in the guinea pig model is associated with cellular immune responses directed against NP and VP35, despite the fact that NP is unable to protect in NHPs (36). The role of GP in mediating protection has been first tested in the lethal challenge studies in guinea pigs immunized with DNA/rAd prime-boost (27), and further data in rodents and NHP suggest that this gene product alone is likely to confer protection (27,45,46).

NATURAL IMMUNITY IN HUMANS Because Ebola outbreaks are sporadic and unpredictable, it has been difficult to analyze the human immune response to natural infection. A variety of retrospective studies have been undertaken in individuals who have survived infection, and associations have been drawn between laboratory analyses and survival (47–56). From such studies, it has been difficult to identify predictive correlates of immunity. After monitoring a large outbreak in Kikwit, Democratic Republic of Congo, in 1995, it was possible to analyze a variety of host immune responses in individuals who were exposed to Ebola virus infection and to correlate outcome with survival (56). An increase in proinflammatory cytokines, including IL-2, interferon-g, IL-10, TNF-a, and interferon-a were found in survivors compared with control subjects, suggesting that increased proinflammatory responses might be associated with an adverse outcome. In survivors of the infection, there was an increase in IgM and IgG levels, particularly to the highly abundant nucleoprotein, which also correlated with the disappearance of viral antigen (54). These data suggested that adaptive immune responses, together with the initial innate response to the infection, may be key to protection against disease. The results from the Gabon survivors suggested that survival or asymptomatic infection could be observed only in individuals who were able to convert their innate immune responses into adaptive immunity. In the Gabon cohort, the activation of T cells early after infection was associated with decreased survival, especially if it was unaccompanied by the ability to generate cytotoxic T cells or in the absence of

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immunoglobulins against viral proteins (47,52,53). Though immunoglobulin against NP was present in survivors and a number of individuals generated antibody responses to VP35 and VP40, it is unclear whether this represents a manifestation of antigen-specific cellular immune responses, or whether these antibodies may have played a more causal role. In any event, such retrospective studies can provide confirmation for immune correlates derived from experimental animal models but are difficult to interpret definitively because of the uncontrolled nature of natural infection.

VACCINE EFFICACY IN NHP Early studies focused on the use of killed vaccines or replication-competent vaccines for both Ebola virus and Marburg. While some degree of efficacy was observed with such vaccines to Ebola and Marburg viruses in guinea pigs (24,57), similar results were not seen for the Ebola virus vaccine in NHPs (58). Because replication-competent viruses pose considerable safety and regulatory concerns, they have not been investigated further. Success in developing protective vaccines in NHPs against other challenging viral diseases such as simian immunodeficiency virus (SIV) has come from gene-based vaccines. Among the vectors that have been analyzed for filoviruses are recombinant DNA, recombinant adenovirus, replication-competent VSV, paramyxoviruses, poxviruses, and VEE replicons. The VEE replicons encoding GP and/or NP from Marburg virus showed protection in cynomolgus monkeys (36), and protection against Marburg virus has been seen with DNA vaccines (unpublished observations). Neither DNA vaccines nor recombinant vaccinia viruses have proven highly efficacious in conferring protection against lethal challenge by Ebola virus (58), in contrast to the experience with Marburg virus in NHPs. The initial success in NHPs came from the use of DNA prime/rAd5 boost with vectors encoding GP and NP (Fig. 1). This immunization strategy was found to elicit both strong cellular and humoral immune responses (40). While a correlation has been demonstrated between survival and the level of ELISA antibody elicited by this vaccine or by a single injection of a rAd vector (41), this protection appears not to correlate with the levels of neutralizing antibody and, on the basis of the murine model, is thought to be dependent on CD8 T-cell responses. For the ELISA antibody response, a threshold titer can be established above which vaccine efficacy is observed (59), and this response correlates with total binding antibodies rather than neutralization. In addition, it has been observed that minor modifications in GP can affect vaccine efficacy, suggesting that the intact GP protein is the most effective immunogen in mediating protection (46). Furthermore, the levels of protection using combinations of GP appear to be uniformly higher and more potent than combinations that included NP in addition to GP (46). The efficacy observed with other modes of immunization, including VSV, paramyxovirus, and other gene-based approaches also appear to require GP, and were associated with T-cell and binding antibody responses (42,43). Taken together, these data suggest that a strong cellular immune response, including CD8 responses, to virus is required for protection. Whether the efficacy can be further augmented in NHPs and humans by effector functions of antibodies remains to be determined; the presence of ELISA binding antibodies correlates with protection when strong CD4 and CD8 responses are achieved.

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Figure 1 Alternative experimental vaccination protocols for first-generation Ebola and Marburg vaccines with (A) DNA prime rAd5 boost or (B) rAd5 immunization only.

POTENTIAL DEVELOPMENT OF HUMAN VACCINES For vaccine studies to advance to licensure for the treatment of human infections, a number of criteria need to be satisfied. First, it will be necessary to demonstrate that the vaccine is similarly immunogenic in humans compared with NHPs. Toward this end, a phase I study of a DNA vaccine has been conducted in humans, which demonstrates the ability to elicit cellular and humoral immune responses to GP and NP constituents of the vaccine (60). The range of antibody titers observed in these studies was similar to those observed in NHPs with a similar regimen (40), suggesting that for DNA vaccines, such a correlation is possible. Secondly, it will be important to demonstrate that the level of cellular and/or humoral immunity that can be achieved in humans and NHPs shows successful protection against lethal challenge in NHPs. It is unethical for human trials to be conducted with Ebola virus challenges, and the occurrence of natural infections is sporadic and not in high enough numbers to be statistically informative. For these reasons, it is likely that licensure of an Ebola virus vaccine will rely on the U.S. Food and Drug Administration animal rule for approval. This mechanism may be used for regulatory review when human clinical efficacy trials cannot be conducted (61). The animal rule represents an alternative pathway for products that have high pathogenicity in humans and rely on animal efficacy studies conducted under good laboratory practices. Both the human safety and immunogenicity information, together with corresponding studies of the same parameters in NHPs, would be required for support of efficacy and approval under 21 CFR parts 314 and 601, described in the Federal Register. In summary, while there is now a considerable body of evidence suggesting that it is possible to protect against lethal viral hemorrhagic fever using gene-based vaccination, the

major challenge at this point is to identify vectors with acceptable safety and efficacy profiles to justify their use. Because it has shown greater responsiveness to a wider range of platforms in NHPs, it is likely that the regulatory requirements will be met for Marburg virus more readily than for Ebola virus. Nonetheless, it would appear that a range of vectors, including DNA, adenovirus, VSV, paramyxoviruses, and possibly viruslike particles, may contribute to the development of a protective vaccine suitable for use in humans. In a recent AIDS vaccine clinical efficacy study, safety concerns regarding the use of Ad5 vectors have been raised because of a possible increase in HIV-1 infections in Ad5-seropositive vaccine recipients (62). At the same time, whether this effect was due to exposure to the adenoviral vector or to other confounding factors in the AIDS vaccine or the study population remains unknown. Because of the concern related to rAd5, alternative adenovirus vectors or combinations of other gene-based vectors may help to achieve

Table 2 Steps Required for Testing and Potential Licensure of an Ebola or Marburg Virus Vaccine . Vaccine immunogen and vector selection – Glycoprotein, DNA, and rAd . Animal efficacy/immunogenicity studies – Rodents – NHPs . Animal safety studies – Rodents, rabbits, and NHPs . Regulatory approvals . IRB/Ethics Committee submissions . Clinical trials and animal efficacy studies (NHPs) in parallel to support licensure under FDA Animal Rule Abbreviations: NHPs, nonhuman primates; rAd, replication-defective adenoviral.

Chapter 80: Development of Vaccines for Ebola and Marburg Viruses

the goal of licensure for Marburg and Ebola virus vaccines. It is likely that a preventive vaccine that would confer long-term immunity in individuals with high-risk and in endemic areas would represent one implementation of such a vaccine strategy, while a second vaccine intended for rapid action after a single injection would be deployed in settings of an acute outbreak. This vaccine approach would be aided by the development of antiviral drugs or monoclonal antibodies that could confer short-term protection while the vaccines elicit the adaptive immune responses required for protection (Table 2).

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19. Lee JE, Fusco ML, Hessell AJ, et al. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature 2008; 454(7201):177–182. 20. Basler CF, Wang X, Muhlberger E, et al. The Ebola virus VP35 protein functions as a type 1 IFN antagonist. Proc Natl Acad Sci U S A 2000; 97(22):12289–12294. 21. Shi W, Huang Y, Sutton-Smith M, et al. A filovirus-unique region of Ebola virus nucleoprotein confers aberrant migration and mediates its incorporation into virions. J Virol 2008; 82(13): 6190–6199. 22. Huang Y, Xu L, Sun Y, et al. The assembly of Ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein. Mol Cell 2002; 10 (2):307–316. 23. Bavari S, Bosio CM, Wiegand E, et al. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses. J Exp Med 2002; 195(5):593–602. 24. Ignatyev GM. Immune response to filovirus infections. Curr Top Microbiol Immunol 1999; 235:205–217. 25. Wilson JA, Bosio CM, Hart MK. Ebola virus: the search for vaccines and treatments. Cell Mol Life Sci 2001; 58(12–13): 1826–1841. 26. Hevey M, Negley D, Geisbert J, et al. Recombinant Marburg virus glycoprotein subunit vaccine protects guinea pigs from lethal infection. In: Brown F, Burton DR, Doherty P, et al. eds. Vaccines 97: Molecular Approaches to the Control of Infectious Diseases. Woodbury: Cold Spring Harbor Laboratory Press, 1997:93–98. 27. Xu L, Sanchez A, Yang Z, et al. Immunization for Ebola virus infection. Nat Med 1998; 4(1):37–42. 28. Pushko P, Bray M, Ludwig GV, et al. Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus. Vaccine 2000; 19(1):142–153. 29. Wilson JA, Hart MK. Protection from Ebola virus mediated by cytotoxic T lymphocytes specific for the viral nucleoprotein. J Virol 2001; 75(6):2660–2664. 30. Bray M. The role of the type I interferon response in the resistance of mice to filovirus infection. J Gen Virol 2001; 82(pt 6):1365–1373. 31. Gupta M, Greer P, Mahanty S, et al. CD8-mediated protection against Ebola virus infection is perforin dependent. J Immunol 2005; 174(7):4198–4202. 32. Gupta M, Mahanty S, Greer P, et al. Persistent infection with ebola virus under conditions of partial immunity. J Virol 2004; 78(2): 958–967. 33. Mahanty S, Gupta M, Paragas J, et al. Protection from lethal infection is determined by innate immune responses in a mouse model of Ebola virus infection. Virology 2003; 312(2):415–424. 34. Wilson JA, Hevey M, Bakken R, et al. Epitopes involved in antibody-mediated protection from Ebola virus. Science 2000; 287(5458):1664–1666. 35. Oswald WB, Geisbert TW, Davis KJ, et al. Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys. PLoS Pathog 2007; 3(1):e9. 36. Hevey M, Negley D, Pushko P, et al. Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology 1998; 251(1):28–37. 37. Jahrling PB, Geisbert J, Swearengen JR, et al. Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses. Arch Virol Suppl 1996; 11: 135–140. 38. Johnson E, Jaax N, White J, et al. Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. Int J Exp Pathol 1995; 76(4):227–236. 39. Ryabchikova EI, Kolesnikova LV, Luchko SV. An analysis of features of pathogenesis in two animal models of Ebola virus infection. J Infect Dis 1999; 179(suppl 1):S199–S202. 40. Sullivan NJ, Sanchez A, Rollin PE, et al. Development of a preventive vaccine for Ebola virus infection in primates. Nature 2000; 408(6812):605–609.

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81 Therapeutics and Vaccines Against Hendra and Nipah Viruses Christopher C. Broder Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland, U.S.A.

INTRODUCTION Hendra virus (HeV) and Nipah virus (NiV) are closely related viral zoonoses that were isolated and subsequently characterized following spillover events associated with significant morbidity and mortality in both animals and humans, and they are the type species of the recently named genus Henipavirus within the family Paramyxoviridae (1). They are enveloped, negativesense RNA viruses, and the family harbors many important human and animal pathogens, including the parainfluenza viruses, Newcastle disease virus, canine distemper virus, measles virus, mumps virus, among others (2). Paramyxovirus-associated pathogenesis is varied, with particular members causing common upper and/or lower respiratory tract infections or less common manifestations of neurological disease. However, HeV and NiV are set apart from all others in the family by their ability to cause a systemic, often fatal, disease in a variety of animals and humans. Large pteropid bats, commonly referred to as flying foxes within the family Pteropodidae, appear to be the predominate natural animal reservoirs for both HeV and NiV on the basis of the observations of both seroconversion prevalence among many species of bats and viral shedding in the absence of detectable clinical disease (3,4). HeV was first recognized in Australia in 1994 in two nearly simultaneous yet unrelated episodes of severe respiratory disease in horses and consequent transmission to three people, two of which were fatal (5). HeV has since reemerged in Australia in 1999 (6), 2004 (7), and 2006–2009, and has always involved horses as an intermediate host along with some additional human infections including two fatalities, the most recent in September 2009, another veterinarian who was infected while conducting a horse necropsy (8,9,109,110). NiV emerged later, first recognized in peninsular Malaysia during a large outbreak of encephalitis among pig farmers in 1998 to 1999 and was primarily transmitted to humans from infected pigs (10). There were 265 cases of human infection, with 105 deaths during this initial outbreak. NiV has continued to reappear, causing at least nine recognized outbreaks in Bangladesh and India since 2001 (5) with the most recent in March 2008 (111). Of particular note, the most recent episodes in 2004 and 2005 were associated with a higher incidence of acute respiratory distress syndrome in conjunction with encephalitis, person-to-person transmission, higher case fatality rates (*75%), and apparently direct transmission from natural reservoirs to humans (11–15, 112).

NiV and HeV are biological safety level-4 (BSL4) restricted and classified as select agents by the National Institute of Allergy and Infectious Diseases (NIAID) and Centers for Disease Control and Prevention (CDC), in category C. However, unlike other notable viral agents of biodefense concern such as smallpox or Ebola virus, they can be isolated from host reservoirs in nature, can be readily grown in cell culture or eggs to high titers (16,17), and can be amplified and spread in livestock serving as sources for transmission to humans, and evidence for nosocomial as well as person-to-person transmission of NiV is possible (11,18,19). There are presently no approved passive or active therapeutic modalities for treating or preventing NiV and HeV infection, and the development of such therapeutics or vaccines too is now of importance.

PATHOGENESIS AND DISEASE MODELS Human Pathology There have been only two fatal of four total cases of HeV infection of humans and both had occurred during the initial outbreak episodes. The first fatal case manifested as a severe respiratory disease, and gross lesions of congestion, hemorrhage and edema associated with chronic alveolitis and evidence of syncytia were noted in the patient’s lungs. The second fatal case occurred in an individual who experienced a brief aseptic meningitic illness after caring for and later assisting at the necropsies of two horses that were later shown to have died from HeV infection. Remarkably, 13 months later, this individual suffered a recurrence of severe encephalitis characterized by uncontrolled focal and generalized epileptic activity, later described as leptomeningitis with foci of necrosis in various parts of the brain parenchyma as well as the presence of endothelial cell syncytia (20). In contrast, the initial NiV outbreak that occurred in Malaysia and Singapore was much larger and autopsies were conducted on 32 individuals revealing immunological and histological features of a systemic endothelial infection accompanied by vasculitis, thrombosis, ischemia, and necrosis (21). Immunohistochemical analyses revealed a widespread presence of NiV antigens in neurons and parenchymal cells within necrotic foci in the central nervous system (CNS) and in endothelial cells of affected blood vessels. Vasculitis and endothelial cell infection was evident in most of the organs examined. Disseminated endothelial cell infection, with vasculitis, thrombosis, and CNS

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infection, all appear to be essential features in fatal human NiV infection (20,21). Although most NiV-infected human patients presented with acute encephalitis, approximately 25% of cases also exhibited respiratory signs, and infection could also present as nonencephalitic or asymptomatic with seroconversion (10). Infection with NiV can also take a more chronic course with neurological disease occurring later (>10 weeks) following a nonencephalitic or asymptomatic infection. The recurrence of neurological disease (relapsed encephalitis) was also observed in patients who previously recovered from acute encephalitis. Relapsed encephalitis presented from several months to as late as two years after the initial infection, and two cases of relapsed encephalitis were observed in 2003, four years after infection (22–24). The underlying mechanisms that allow these viruses to escape immunological clearance for such an extended period are completely unknown and uniquely fascinating (1).

Cellular Tropism and Host Range In addition to their environmental hosts, flying foxes, NiV is known to infect pigs, horses, cats, dogs, and man, and experimental infections of guinea pigs, hamsters, and cats have been demonstrated. On the other hand, HeV appears to be less transmissible in the environment, and no naturally acquired infections other than those observed in bats, horses, and human have been described; however, experimentally HeV will infect guinea pigs and cats (5,25). These natural and experimental infections of animals also correlate with HeV- and NiVmediated cell fusion assays conducted in vitro (26,27). Together, these observations highlighted the unusually broad species tropism and potential host range of HeV and NiV. HeV and NiV possess two glycoproteins anchored in their membrane, one associated with attachment and designated G because it has neither hemagglutinating nor neuraminidase activities, features that are associated with many other paramyxovirus attachment glycoproteins known as hemagglutininneuraminidase (HN) or the hemagglutinin (H). The second is the fusion (F) glycoprotein, which directly facilitates the fusion of the viral and host cell membranes at neutral pH resulting in infection of the cell (2,28). Cells expressing these glycoproteins on their surfaces can also fuse with other receptor-bearing cells, resulting in the formation of multinucleated giant cells (syncytia), a hallmark of their cytopathic effect (CPE). Whereas the majority of well-characterized paramyxoviruses possess neuraminidase activity and employ sialic acid moieties as receptors, some use cell-surface proteins as receptors such as measles virus and canine distemper virus, which utilize CD46 and SLAM (29–32). It was speculated early on that HeV and NiV likely also employed a cell-surface protein(s) as a receptor as well. Subsequently, ephrinB2 was identified as the receptor employed by NiV (33,34) and for HeV as well (33). Also, ephrinB3, a related protein with some significant sequence homology, has also been shown to be a functional receptor for both (34,35). EphrinB2 and B3 are members of a family of surface expressed glycoprotein ligands that bind to Eph receptors, the largest subgroup of receptor tyrosine kinases (36–38). The Eph receptors and their ephrin ligands make up an important group of bi-directional signaling molecules known to participate in many instances of cell-cell interactions, including those of vascular endothelial cells and are modulators of cell remodeling events, especially in the CNS. EphrinB2 is expressed in neurons, smooth muscle, arterial endothelial

cells, and capillaries (39–41) and its identification as a receptor for HeV and NiV has helped clarify their broad tissue tropism and consequent pathogenic processes.

Animal Pathology and Potential Disease Models The development and characterization of suitable animal models to study NiV and HeV infection is essential for understanding their pathogenic features as well as fulfilling a critical requirement for the evaluation of antiviral therapeutics and vaccines. Presently, there is a paucity of accepted animal models for NiV or HeV infection, and the restriction of live virus experimentation to BSL4 containment has significantly hampered a rapid and systematic approach toward their development. Further, the U.S. Food and Drug Administration (FDA) has implemented the Animal Efficacy Rule, which came into effect in 2002, which specifically applies to the development of therapeutic products when human efficacy studies are not feasible or ethical, such as in the case of highly virulent emerging pathogens. Essentially, with this regulation, licensure can be granted on the basis of animal studies that bridge to human immunogenicity and safety studies to evaluate product efficacy in treating or preventing disease caused by a specific agent. Such approval required a well-understood mechanism for the pathogenicity of the agent, the mechanism of protection by the product, and the demonstration of its effectiveness in more than one animal species. The initial cases of HeV in Australia involved horses, and NiV was associated with pigs in Malaysia, although dogs, cats, and horses were also involved (5). The pathology caused by either virus in horses is of greater severity than that caused by NiV in pigs. Infectivity and transmissibility experiments with HeV have been carried out in horses, cats, and bats (42). The incubation period in naturally infected horses appears to be between 8 and 11 days. Initially, horses become anorexic and depressed with general uneasiness and ataxia, and animals develop a fever with sweating. Respiration becomes rapid, shallow, and labored; there is congestion with a frothy yellow nasal discharge noted as a common terminal feature between one and three days following the onset of clinical signs. Cats inoculated with HeV develop clinical disease and shed virus in urine, but cats suspended in cages above fed troughs used by infected horses did not become infected. However, infected cats in similar cages transmitted infection to one of three contact horses, and cat-to-cat transmission was seen in animals sharing cages (43). Infected cats become depressed, with fever and elevated respiratory rates, and there is rapid progression to severe illness and death within 24 hours of the onset of clinical signs (44,45). NiV infection of pigs has also revealed the respiratory system as a primary target organ of virus replication and pathology, with viral antigen present in the respiratory epithelium (tracheal, bronchial, bronchiolar, and alveolar), including intranasal epithelial cells (46) (Table 1). Virus was also evident in the kidneys and endothelial and smooth muscle cells of blood vessels, and endothelial cells of lymphatic vessels. The involvement of the CNS was more rare, but virus was observed in arachnoid cells of the meninges and in the connective tissue surrounding the trigeminal ganglion (46). More recently, a piglet model of NiV infection has been explored, in which five-week-old piglets were infected intranasally, orally, and ocularly with a larger amount of virus (2.5  105 PFU [plaque forming unit]) and then euthanized at three and eight days post

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Table 1 Animal Models of Hendra Virus and Nipah Virus Infection Comparison of Hendra virus pathology in animals and humansa

Comparison of Nipah virus pathology in animals and humansb ND

Pigd

Similarities Systemic vascular disease. Endothelial syncytia evident; gross signs of pneumonia. Virus in kidney and in urine; (virus in human kidney, urine not tested). Differences Clinical response frequently mild and variable from unapparent to sudden death. Virus targets larger vessels in preference to capillaries. Viral antigen located preferentially in tunica media. Severe pulmonary edema not evident. Only a proportion of animals develop encephalitis. ND

Hamstere

ND

Catf

Similarities Systemic vascular disease severe effects in the lungs. Endothelial syncytia evident. Virus in kidney and in urine; (virus in human kidney, urine not tested).

Animal Guinea pigc

Differences Virus present in brain but no lesions found.

Differences Transient fever and weight loss with full recovery.

Similarities Systemic vascular disease. Differences Respiratory system a primary organ target. Although evident, CNS involvement was rarer. No virus in urine. Similarities Brain severely affected with vascular and parenchymal lesions. Endothelial syncytia evident. Viral antigen and large inclusion bodies were apparent in neurons. Virus shed in urine. Differences Absence of parenchymal necrosis in the lung, presence of vasculitis in the liver. No pulmonary edema in lung. Similarities Systemic vascular disease, severe effects in the lungs. Endothelial syncytia evident. Extensive inflammation in the respiratory epithelium associated with viral antigen. Virus shed in urine. Differences Virus detected in CNS but no lesions noted.

Key references and not meant to be exhaustive. a From Refs. 106 and 107. b From Refs. 14, 21, and 108. c From Refs. 49, 53, and 54. d From Refs. 46 and 47. e From Ref. 49. f From Refs. 44, 48, 50, and 53.

infection (47). Consistent with infected pigs during the Malaysian outbreak, the virus caused neurological signs in only 2 of 11 animals, while the rest remained clinically healthy. Evidence of virus replication and dissemination was widespread in the animals, with significant amounts detected in the respiratory system. Notably, the presence of high levels of virus was confirmed in the CNS of sick as well as apparently healthy animals. Recoverable virus was obtained from the respiratory, lymphatic, and nervous systems (47). The clinical and pathological syndrome induced by NiV has also been explored in cats and is similar to that associated with HeV but with more extensive inflammation of the respiratory epithelium, associated with viral antigen (Table 1). In the initial NiV infections of the cat by oronasal challenge with a 50,000 tissue culture infectious dose (TCID)50, gross lesions in those animals with severe clinical signs consisted of hydrothorax, edema in the lungs, and pulmonary lymph nodes,

froth in the bronchi, and dense purple-red consolidation in the lung (46). There were also similar histological features, including diffuse perivascular, peribronchial, and alveolar hemorrhage and edema, vasculitis affecting arteries and arterioles, and alveolitis with syncytia among endothelial and alveolar epithelial cells (20). More extensive experiments with NiV in cats have shown that animals succumb to subcutaneous infection with doses as low as 500 TCID 50 (48) typically within six to eight days. NiV disease in cats presents as an acute febrile reaction accompanied by subtle changes in behavior, followed by severe respiratory disease along with an underlying systemic vasculitis. A Taqman polymerase chain reaction (PCR) assay was developed enabling the detection and quantitation of viral RNA and assessment of the levels and locations of virus. As expected, the lungs were a primary site of viral replication, consistent with the extensive pulmonary pathology. Additionally, lymphoid tissues (lymph nodes

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and spleen) and highly vascularized tissues displayed high relative quantities of viral genome. The systemic vasculitis seen in the cat model is consistent with the resulting pathology of NiV infection that has been observed in all known susceptible animals, including humans (20,21,46,49). In utero transmission of NiV in cats has also been recently demonstrated (50) with evidence of extensive viral replication in many tissues of a pregnant adult cat and in fetal tissues, suggesting both vertical and horizontal transmission of this virus is possible, having implications for the potential transmission of NiV infections (50). The importance of pregnancy and fetal materials in disease spread was first hypothesized after the discovery that the index case of the 1994 HeV outbreak was a pregnant mare (51) and vertical transmission of HeV was later experimentally confirmed in guinea pigs and bats (52). For practical reasons, smaller animal models have been explored for both HeV and NiV infection. Guinea pigs have been experimentally infected with HeV; however, the pathology seen in the guinea pig differed significantly in several respects compared with human cases and naturally and experimentally infected horses (53,54). The response to HeV by guinea pigs has also been variable, ranging from frequently mild to sudden death. In guinea pigs, HeV causes a generalized vascular disease but, unlike horses and cats, there is little or no pulmonary edema (Table 1). Nevertheless, histologically, vascular disease is prominent in arteries and veins, and in many organs. NiV infection of the guinea pig has yet to be well described, but one report demonstrated that guinea pigs infected by the intraperitoneal route with 107 PFU of NiV showed only a transient fever with minor weight loss after five to seven days but later recovered (49) (Table 1). The use of typical, small animal models has been problematic; HeV and NiV do not cause disease in mice following a subcutaneous challenge (Crameri and Eaton, personal communication) or with either an intranasal (6  105 PFU) or intraperitoneal challenge (107 PFU) of NiV (49). In addition, there is neither serological evidence of HeV infection among a variety of rodent species from Australia (55) nor any evidence of NiV infection in rodents from Malaysia (10,56). The susceptibility of rabbits to infection and disease has been explored only with HeV, and no clinical disease was observed (45). The only small animal model that has proven useful thus far has been the golden hamster with a NiV challenge (49) (Table 1). Hamsters infected by an intranasal (6  105 PFU) or intraperitoneal route (107 PFU) with NiV demonstrated that infection by either challenge route was manifested as difficulties in movement and balance in the animals, which then rapidly died in five to eight days. RT-PCR demonstrated viral genome in the urine, heart, liver, spleen, kidney, lung, brain, and spinal cord with the intraperitoneal route of infection yielding a more rapid and consistent disease course with more tissues positive for genome and cultivatable virus. Histopathological examination revealed that blood vessels, especially in the CNS, developed vasculitis characterized by necrosis and inflammation with involvement of the vessel wall, including endothelial and smooth muscle cells, revealing the presence of antigen, genome, and syncytia. There were some notable distinctions between the overall pathology in the hamsters versus that seen in humans. In the lung, areas of parenchymal inflammation with vasculitis were less evident and pulmonary edema and syncytia were lacking. Severe pathological lesions were most evident in the brain, while thrombosis and vasculitis and syncytia were seen in the blood vessels of multiple organs (49). Overall, both the golden

hamster and cat represent viable models that can be used to examine alternative aspects of disease caused by either HeV or NiV. Table 1 summarizes and compares these animal models.

THERAPEUTIC AND VACCINE STRATEGIES The present chapter reviews the current focus of research on the development of potential vaccines and therapeutic modalities for HeV and NiV; however, one well-known antiviral drug that has actually been used in the treatment of NiV infection (ribavirin) bears mention here. Ribavirin (57) is likely the bestknown, first-line therapeutic for suspected viral diseases of unknown etiology. It exhibits antiviral activity against a wide variety of both RNA and some DNA viruses. It is an accepted or approved treatment for several viral infections, including RSV, arenaviral hemorrhagic-fevers, particularly Lassa fever virus and some bunyaviruses (58). Because of its global commercial availability, its off-label use is often employed for the treatment of viral diseases under conditions where supportive care is the only alternative. While there is some evidence that ribavirin therapy may be of clinical benefit (59), recent experimental infection models have not supported this (60).

Viral Envelope Glycoproteins For HeV and NiV, the development of potential vaccines and therapeutics has focused largely on targeting virus attachment and infection, processes facilitated by the viral envelope glycoprotein spikes. Nearly all potential antiviral products that have been reported on to date have targeted the viral glycoproteins. As discussed earlier, attachment and membrane fusion is facilitated by the G and F glycoproteins. G is a type II membrane glycoprotein: its NH2-terminus is oriented toward the cytoplasm and its COOH-terminus is extracellular, consisting of a stem (or stalk) and globular head structure (28). The structure of the Henipavirus G has yet to be determined; however, both HeV and NiV G can accommodate a six-bladed b-propeller structural model similar to other paramyxovirus H or HN glycoproteins (61,62). The other envelope glycoprotein is F, and it is directly involved in mediating fusion between the virus and host cell membranes. F is a type I membrane glycoprotein with the extracellular NH2-terminus composed of several features conserved in other viral fusion proteins, such as gp120/gp41 of human immunodeficiency virus type 1 (HIV-1), and the hemagglutinin (HA) of influenza virus (63). Biologically active F consists of two disulfide-linked subunits, F1 and F2, which are generated by the proteolytic cleavage of an F0 precursor. The membrane-anchored subunit, F1, contains a new hydrophobic NH2-terminus referred to as the fusion peptide. With few exceptions, all paramyxoviruses require both an attachment glycoprotein and F for efficient fusion, but a complete understanding of the mechanism(s) by which these glycoproteins mediate membrane fusion remains unknown (28). F glycoproteins exhibit class I fusion, involving two a-helical domains known as heptad repeats that mediate the formation of a trimer-of-hairpins or six-helix bundle (6-HB) during or immediately following fusion. Peptides corresponding to either heptad repeat can potently inhibit the fusion activity when present during the process of membrane fusion (28). In addition, all viral envelope glycoproteins are homo- or hetero-oligomers in their mature and functional forms (64) and such multimeric proteins will often have structural differences between their monomeric subunits versus the mature oligomer.

Chapter 81: Therapeutics and Vaccines Against Hendra and Nipah Viruses

This feature can also translate into differences in antigenic structure and has been shown for a number of proteins, most notably with influenza HA (65) and HIV-1 gp120/gp41 (66). All paramyxovirus, retrovirus, and influenza virus fusion proteins exist as homotrimers and most recently both post- and prefusion paramyxovirus F glycoprotein structures have been determined (67–69) (28). Paramyxovirus attachment glycoproteins such as H of measles virus (70), several HN glycoproteins, and the Henipavirus G glycoproteins have all been shown to exist as disulfide-linked dimers that can associate into tetrameric forms (28). Below, we will review several passive and active therapeutic or vaccination modalities that have been explored as intervention strategies for HeV and NiV infection; but we will limit the discussion to those that have been advanced the farthest to date and target the viral envelope glycoproteins.

Passive Therapies Fusion Inhibitors The first potential Henipavirus-specific therapeutic was shown to be a heptad peptide-based fusion inhibitor (26). As discussed above, peptides, typically 30 to 40 residues in length that are homologous to either of the heptad repeat domains of several paramyxovirus F glycoproteins, including HeV and NiV, have been shown to be inhibitors of membrane fusion by preventing the formation of the 6-HB structure (28,71). Targeting this initial membrane fusion step of the viral infection process has been the focus of much attention brought on by the initial observations on HIV-1 (72). Indeed, the HIV-1-specific peptide, enfuvirtide (FuzeonTM, Roche, New Jersey, U.S.), which was approved by the FDA in March 2003 is a 36-amino acid peptide corresponding to a portion of the COOH-terminal heptad repeat of gp41, and it has proven clinically successful, allowing for expanded optimized combination therapies (73,74). It has been shown that peptides derived from the COOH-terminal heptad repeat of either HeV or NiV F were potent inhibitors of fusion as well as live virus infection with IC50 concentrations in the nM range (75,76). In fact, a single peptide sequence possessed sufficient homology to inhibit both HeV and NiV membrane fusion, and these peptides are presently being evaluated in vivo using the NiV infection cat model (Mungall and Broder, unpublished). It is anticipated that fusion inhibitors would reduce the systemic spread of the virus, dampening replication and reducing its pathogenicity, thus affording the host time to generate an effective immune response. Antibodies It is almost without exception that all neutralizing antibodies to enveloped viruses are directed against the virus’ envelope glycoproteins, which project from the surface of the virion particle. Traditionally, the antibody response has been the immunological measure of vaccine efficacy. While neutralizing antibodies elicited by a vaccine can be highly effective, they can also be administered passively to acutely infected individuals and can often be equally efficacious. The mechanism of passively administered antiviral antibody therapy could be viewed as that of an antiviral drug, suppressing ongoing infection permitting the host to mount an effective and often sterilizing immune response. Today, passively administered antibody is routinely used as an effective antiviral therapy or prophylactic for hepatitis B, varicella-zoster, rabies virus, and RSV among others (77). In most cases, their use is a first-line therapy as a postexposure measure or in circumstances where vaccination is

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not possible. However, serum polyclonal antibody preparations have associated problems related to toxicity and potential allergic reactions, as well as lot-to-lot variation and uncertain dosing regimes (78). For paramyxoviruses, antibodies specific for either the attachment or F glycoproteins can neutralize virus; however the preponderance of data indicate that those antibodies directed against attachment glycoproteins, appear to be the dominant target antigen for neutralizing antibodies (79–81). The first evidence of passive protection against a NiV challenge was demonstrated using hamsters (82). Here, various pools of monospecific polyclonal antiserums against F and G were prepared using a prime-boost immunization strategy by infecting hamsters with recombinant vaccinia viruses encoding either F or G and later boosted with lysates of BHK21 hamster cells expressing F or G by vaccinia virus prepared in complete Freund’s adjuvant. Control antisera was also prepared using wild-type vaccinia virus. The protective efficacy of the various antisera to either G or F or a mixture of both were tested using the hamster model (Table 2) (49). The animals were given 200 mL of one of the antisera followed one hour later by challenge virus, and at 24 hours another 200 mL of antisera was administered, all by intraperitoneal injection. On the basis Table 2 Successful Passive and Active Immunization Strategies for Nipah and Hendra Viruses Strategy Passive immunization Polyclonal antibodya Monoclonal antibodiesb Active immunization Recombinant vaccinia virusc Recombinant canarypox virusd Subunite

Viral antigen

Animal model used

Nipah F and/or G glycoproteins Nipah F and/or G glycoproteins

Golden hamster Golden hamster

Nipah F and/or G glycoprotein Nipah F and/or G glycoprotein Nipah or Hendra soluble G glycoprotein

Golden hamster Pig Cat

a Polyclonal monospecific serum against Nipah F and G glycoproteins were prepared using a prime-boost immunization strategy by infecting hamsters with recombinant vaccinia viruses encoding either F or G followed by inoculation with lysates of BHK21 cells infected with F or G encoding recombinant vaccinia virus in complete Freund’s adjuvant. The protective efficacy of these antisera was tested by intraperitoneal administration of hamsters followed by an intraperitoneal challenge with Nipah virus (82). b Two murine monoclonal antibodies against Nipah F and two against G glycoproteins were examined for their protective efficacy as antibodycontaining ascitic fluid by intraperitoneal administration into hamsters before and following an intraperitoneal challenge with Nipah virus (88). c Nipah virus F and or G encoding recombinant vaccinia viruses were used to immunize hamsters twice with a month interval. 107 PFU of either the F or G encoding recombinants were used or 5  106 of each in combination. The animals were challenged by an intraperitoneal injection of Nipah virus (82). d Nipah virus F and or G encoding recombinant canarypox viruses were used to immunize four-week-old pigs twice with a two-week interval. 108 PFU of either the F or G recombinants were used or 108 of both in combination. The animals were challenged intranasally with 2.5  105 PFU total split between each nostril 28 days post vaccination (97). e A purified, soluble form of the G glycoprotein (sG) derived from Nipah virus or Hendra virus was used to immunize cats. Three 100-mg doses of either Hendra virus sG or Nipah virus sG in CSIRO triple adjuvant [60% (vol/vol) Montanide, 40% (vol/vol) sG (combined with Quil A, 3 mg/mL, and DEAEdextran, 30 mg/mL) in water] was administered by subcutaneous inoculation, at two-week intervals (48).

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of prior studies, the 1000 PFU challenge of virus was approximately 3.7 LD50 (49), and in this study the anti-F and anti-G, as well as the combination of the two, were all shown protective. Given the large dose, location, and timing of the NiV-specific neutralizing serum used, combined with the challenge dose of virus, the protection afforded by this passive immunization protocol may not be unexpected. Nevertheless, the study demonstrated the importance of the humoral response to the viral envelope glycoproteins of NiV. Although specific polyclonal antiserums can be effective, the development of monoclonal antibodies (mAbs) including chimeric animal-human or humanized mAbs has made passive antibody therapy development a major focus of current research (83,84). For example, a humanized mAb to RSV F Synagis1 (palivizumab) (MedImmune, Inc. Gaithersburg, MD) is a more cost-effective and efficacious treatment than the original polyclonal product (85) and is the only mAb against a viral disease approved by the FDA. However, it is used prophylactically and is not an effective treatment for established infections (86), but recently Palivizumab has been improved and motavizumab has been shown to very potently inhibit viral replication in the upper respiratory tract in a cotton rat model (87). A follow-up study using two murine mAbs against NiV F and two against G as passive immunotherapies has also been performed (88). Here, the mAbs were examined as antibody in ascitic fluid delivered to hamsters by intraperitoneal administration. A series of experiments were conducted with differing amounts of mAbs administered 24 hours before virus challenge and again at 1 hour following virus challenge, using 100 LD50 of NiV. In addition, the efficacy of either anti-F or anti-G mAbs from 1 to 96 hours following virus challenge was also evaluated. The animals that received the mAbs in sufficient amounts before and immediately following the intraperitoneal challenge of NiV were completely protected (Table 2) (88). These mAbs were exceptionally potent, and only 1.2 mg of an anti-G mAb could completely protect the challenged animals under these conditions. As before, although F-specific antibody could also afford protection, more anti-F mAb in comparison with anti-G was required. Again, in light of the earlier findings with polyclonal sera and because of the route and timing of the administration of these highly potent neutralizing mAbs and virus dose, protection was not unexpected. High levels of either anti-G or anti-F mAbs appeared to yield sterilizing immunity, while lower amounts of antibody could still protect against fatal infection but did result in measurable increases in antiNiV antibodies following virus challenge. Nevertheless, these studies support the notion that passively immunotherapy directed against the viral envelope glycoproteins as a viable therapeutic modality for treating NiV infection is possible. However, a passive immunization experiment using antibody administered systemically before or following virus challenge has yet to be evaluated, and the humanization of these murine mAbs will take considerable effort and time to formulate an acceptable therapeutic product. A major advance in the development of specific mAbs has been the use of bacteriophage display of combinatorial antibody libraries (89–90). The phage libraries can be prepared to encode human antibodies as Fab fragments or single-chain variable region fragments (scFvs), and this technology has been complemented by innovative affinity maturation strategies to improve antibody binding characteristics (91). These new techniques in human phage display antibody platforms have facilitated the rapid identification and isolation of specific human

mAbs, eliminating the laborious processes of immunization, hybridoma development, and the humanization process. Using these techniques, neutralizing human mAbs reactive to HeV and NiV G have been identified, isolated, and characterized (92). These mAbs were generated by panning a large naı¨ve human phage display antibody library containing about 1010 different phage-displayed Fabs using a recombinant, soluble G glycoprotein (sG) derived from HeV. In particular, two Fabs, m101 and m102, had significant neutralizing activities against live virus and m101 was converted to a full-length human IgG1 antibody. IgG1 m101 was exceptionally potent in neutralizing infectious HeV; complete (100%) neutralization was achieved with 12.5 mg/mL and 98% neutralization with 1.6 mg/mL using a 96-well plate based assay using 200 TCID50 of virus with Vero cells. Because of its potent cross-reactivity to both HeV and NiV G, the mAb m102 has been affinity matured and converted to a full-length human IgG1 antibody (m102.4). Examination of the antibody as an inhibitor of HeV and NiV cell fusion revealed potent inhibitory activity. IgG1 (m102.4) can block live HeV and NiV infection with 90% neutralization requiring less than 0.5 mg/mL (113). These are the first fully human mAbs identified against these viruses and considering their potency in vitro, they could provide a valuable postexposure or postinfection therapeutic modality for treating NiV or HeV infection.

Active Vaccines While it is not clear which arm of the immune system is the more important in defense against viral pathogens, antibodies have been shown effective in neutralizing NiV and HeV, and neutralizing antibodies are the key vaccine-induced protective mechanisms in the case of some well-known paramyxovirus human pathogens, mumps and measles viruses (93,94). Indeed, all successful human viral vaccines induce neutralizing antibodies that can cross-react with immunologically relevant strains of a given virus (95). Live Recombinant Vaccines There are presently 15 viral vaccines approved for human use, excluding various viral subtypes, and the majority of these are live-attenuated formulations (96). Two highly effective examples are those developed for the paramyxoviruses, mumps, and measles viruses. In general terms, these live-attenuated viral vaccines are highly effective because they elicit a balanced immune response in the recipient host, stimulating both cellmediated and humoral immunity. However, because of the highly lethality of HeV and NiV, it is unlikely that live-attenuated versions of these agents would be approved as vaccines for use in humans. Alternatively, live recombinant vaccines and varied platforms are currently under consideration (96). The first vaccination and challenge protocol for NiV was carried out in the hamster model using a recombinant vaccinia virus–based platform (82). Here, NiV F and G encoding recombinant vaccinia viruses were examined individually and together by immunizing hamsters twice with a one-month interval, using 107 PFU of either the F or G encoding recombinants or 5  106 of each recombinant together. The recombinant full-length glycoproteins, expressed by vaccinia virus were evaluated for cell-surface expression as well as function using a syncytia formation assay in cell culture. Antibody titers measured by ELISA and virus neutralization following the booster immunization were modest with the sera raised against

Chapter 81: Therapeutics and Vaccines Against Hendra and Nipah Viruses

the NiV G recombinant eliciting the strongest response (*1:4000 and <1:50, respectively). Nevertheless, all animals were completely protected following an intraperitoneal challenge of 1000 PFU of NiV, whether immunized with the G or F or both vaccinia virus recombinants (82). Both ELISA and neutralizing antibody titers against NiV rose considerably following virus challenge indication virus replication. Although the contribution of cell-mediated immunity to protection in this experiment cannot be excluded, passive transfer experiments with antisera prepared using these vaccinia recombinants demonstrated protection (discussed earlier) indicating major roles for specific antibody. Although a highly attenuated vaccinia virus strain, NYVAC, was employed, this platform as a recombinant vaccine for humans is unlikely. The development of potential livestock vaccines for HeV and NiV may also be desirable, and recently, a recombinant canarypox-based vaccine candidate for swine has been explored (97). Similar to the vaccinia virus constructs described above, the NiV F and G genes were used to generate recombinant canarypox virus (ALVAC) vaccine vectors. These recombinant viruses were used to immunize four-week-old pigs twice with a two-week interval. Similar to the other study, each recombinant was tested alone and in combination, and 108 PFU of either the F or G recombinant were employed or a 108 dose of each. The piglets were challenged intranasally with a 2.5  105 PFU dose, which was divided between each nostril at 28 days post vaccination. As described previously, NiV infection of pigs is a much milder disease with neurological signs in only a small percentage of animals, while most remain clinically healthy. However, the virus does replicate and disseminate to a variety of organ systems with significant levels of recoverable virus present in the respiratory system. The goal of this vaccination study was twofold, to prevent disease and to impede virus shedding. The results of the study demonstrated protection from NiV-associated disease in all vaccinated animals by the G, F, or both ALVAC vectors. In addition, only low levels of viral RNA were detectable and in only a few tissue samples and no isolatable virus was observed in the vaccine recipients. In contrast, both high levels of viral RNA as well as isolatable virus were consistently obtained in the control-challenged pigs, notably in the throat and nose. Neutralizing antibody was elicited by both vectors, with ALVAC-G yielding the strongest response approximately fivefold higher (*1:1280). The combined ALVAC-F/G vaccination appeared to be only marginally better than that of G alone, and the data indicated that either formulation could serve as a protective vaccine against NiV for swine (97). Subunit Vaccines Given the highly pathogenic characteristics of HeV and NiV, the development of recombinant subunit immunogens also represent a viable avenue for vaccine development because they are extremely safe and can be administered with no risk of infection. Recombinant, soluble versions of G (sG) from both HeV and NiV have been developed as potential subunit vaccines (98). sG has been shown to retain many important structural, functional, and antigenic properties similar to native full-length G, including retention of oligomeric structure, the ability to bind virus receptor (ephrinB2 and B3) and block membrane fusion and infection, and elicit strong polyclonalneutralizing antibody responses in rabbits, mice, and cats. Further, sG was also used as an antigen for the panning and isolation of neutralizing human mAbs, discussed above (92).

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Most recently, these sG preparations have been used in the development of a Bio-Plex Luminex1 platform assay that can simultaneously detect and differentiate HeV- and NiV-specific neutralizing antibodies in serum in the absence of live virus (99). Given the biological features possessed by recombinant sG, it would seem an ideal subunit vaccine candidate. Consideration should also be given to the protein sequence homology among the Henipavirus G glycoproteins, which will also affect the antigenic relatedness of one G species to another. Previous genomic sequencing and analysis revealed that HeV isolates obtained from infected horses and the fatal human case during the outbreak in 1994 were essentially identical to those obtained from flying foxes two years later from the same area (4,51). Whereas NiV is more diverse, and several distinct isolates have been described and there appears to be at least three distinct lineages of NiV when the isolates from Malaysia (3,100,101), Bangladesh (102), and Cambodia (103) are compared (1). The pathogenic characteristics of these different variants in human and animal hosts are apparently different as well. Regardless, for HeV and NiV vaccines that stimulate the production of neutralizing antibody responses targeting the viral envelope glycoproteins, these species and strain differences do not appear to be problematic on the basis of recent heterologous immunization and challenge experiments (discussed below) (48). The HeV and NiV F share an 88% amino acid identity along with the conservation of six potential N-linked glycosylation sites in their extracellular domain, while the G glycoproteins exhibit 83% amino acid identity with conservation of seven extracellular N-linked glycosylation sites (104,105). By comparison, among the lineages and strains of NiV, the percent amino acid identity between the various G and F glycoproteins is even greater and on the order of 95% to 98%, respectively (1). In initial experiments, the immunization of rabbits with HeV sG yielded a cross-reactive, neutralizing anti-G antiserum that yielded complete neutralization of HeV at a dilution of 1:1280 and NiV at a dilution of 1:640 (98). This twofold difference in neutralization titer was consistent with the partial antibody cross-reactivity of HeV and NiV G, and earlier studies also demonstrated that HeV- and NiV-specific antisera do crossneutralize (16). Because HeV sG was able to elicit such a potent neutralizing antibody response, its use as a possible subunit vaccine was subsequently explored using the cat model (Table 1). Four cats were immunized with three 100-mg doses of sG; two animals received HeV sG, and two received NiV sG formulated in CSIRO triple adjuvant and administered by subcutaneous inoculation, at two-week intervals (48). Two weeks after the immunization protocol, all the vaccinated animals exhibited exceedingly high homologous serum neutralizing titers (1:20,480). Notably, the animals immunized with HeV sG had similar heterologous neutralizing titers (1:20,480), whereas NiV sG-immunized cats exhibited somewhat lower heterologous neutralizing titers ranging from 1: 1280 to 1:2560. The retesting of serum at two months revealed no decrease in homologous neutralizing titers, while there was a two to eightfold drop in heterologous titers. The four immunized cats and two additional naı¨ve controls were challenged subcutaneously with 500 TCID50 NiV approximately two months after the third immunization. Both control animals developed lethal disease 6 to 13 days post challenge, while none of the immunized animals showed any sign of disease. Taqman PCR analysis of samples from control animals revealed considerable levels of

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NiV genome in a wide range of tissues, while genome was detected in only two immunized cats in only four samples, but below the limit of accurate detection (48). Although the numbers of immunized animals were minimal and despite the lower level of heterologous neutralizing antibody titers, both immunogens could completely protect against subsequent NiV challenge, suggesting that a single vaccine may be effective against both viruses, with HeV sG eliciting the more potent cross-reactive antibody response. Indeed, Bossart et al. has recently analyzed antibody responses in sera from naturally infected or immunized sources and has shown that although the specificity of antibody responses to G mirrored the virus that elicited the response, HeV-infected individuals had high levels of NiV G cross-reactive antibodies; whereas NiV-infected individuals had limited cross-reactive antibodies to HeV G (99). Together, these data suggested that the native HeV G stimulates a more robust and cross-reactive immune response, similar to these immunization data.

SUMMARY AND CONCLUSIONS HeV and NiV remain the first and only examples of zoonotic paramyxoviruses that can infect and cause lethal disease across a broad range of vertebrate hosts. Presently, there are no approved treatment modalities for humans or animals. Because of the environmental availability of these agents, their associated high morbidity and mortality and the risks from natural infections, laboratory accidents, or their potential deliberate misuse, the development of effective countermeasures is now a priority. Much of the research conducted over the past several years has centered on the virus binding and entry, including the attachment to receptors and membrane fusion. These efforts have led to the development and preliminary testing of both potential vaccine candidates and antiviral therapeutics, with the commonality of targeting these very early stages of the infection process; however, significant challenges remain. Typically, because of the complexity of conducting animal experiments under high level bio-containment and the physical demands on the staff involved, only limited numbers of virus challenge experiments are feasible at any single facility. In addition, there are only a limited number of suitable animal models of Henipavirus infection available. At present, the hamster and cat models of NiV infection have been examined in the greatest detail, but neither is yet recognized as a gold standard, and further research is needed. Nevertheless, there exists the requirement for an acceptable animal model in evaluating the efficacy of new antiviral products or vaccines for viral agents such as HeV or NiV. Importantly, a nonhuman primate model of Henipavirus infection has yet to be described but will likely be a future requirement, and though costly, such experiments are nonetheless feasible and should facilitate significant developments toward effective treatments of NiV and HeV infection.

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82 Vaccines Against Lassa Fever Maria S. Salvato and Igor S. Lukashevich Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION A 2004 review of viral hemorrhagic fevers by Geisbert and Jahrling considered a Lassa vaccine to be ‘‘low-hanging fruit,’’ meaning that it should be an easily attainable goal for vaccinologists (1). They assessed the promising vaccine candidates at that time. Groups in the United States and the United Kingdom had already showed that vaccines could protect guinea pigs if they expressed either the nucleocapsid protein (NP) or the envelope glycoprotein of the Lassa virus. The nonhuman primate model was more discriminating than the guinea pig model, but it too could be protected by a vector that only expressed the envelope glycoprotein of Lassa virus. Lassa outbreaks have caused twice as many deaths as Ebola outbreaks. Lassa outbreaks have increased during political upheavals in West Africa, such as the 1999 to 2005 conflicts in Sierra Leone and Liberia. Also, travel of individuals from West Africa has occasionally brought Lassa virus outside the endemic area; for example, a single-source outbreak in September 2008 afflicted five people in South Africa, where Lassa is not endemic. Since seroepidemiologic studies in endemic areas suggest the occurrence of subclinical and mild infections, in addition to more severe clinical cases, expectations have been raised that resistance could be attained by vaccination. Despite promising results from a number of experimental vaccines in animal models, the performance of clinical trials remains a hurdle. Lassa virus was already classified as a riskgroup 4 agent to be propagated only at biosafety level 4, and the terrorist attack on New York City on September11, 2001, led to its classification as a category A pathogen (2), meaning it became high priority for biodefense. As a consequence of the infusion of biodefense funding, several experimental Lassa vaccines were introduced (3–8), and one, ML29, succeeded in meeting the FDA ‘‘two-animal rule’’ for promising tests in animal models (3–5). Here we will review the characteristics of Lassa virus infection, vaccine strategies, and some of the experimental vaccines. We will mention the potential for postexposure vaccination for prevention of Lassa fever, and will review the use of new technology in standardizing these vaccines.

BACKGROUND ON LASSA VIRUS The Arenavirus Family Arenaviruses are rodent-borne, negative-stranded RNA viruses that have been recognized as emerging human pathogens (9). Several New World arenaviruses have been described in South America (10–16). In the United States, arenaviruses caused a

hepatitis outbreak in zoo monkeys (17,18) and Whitewater Arroyo virus was found in southwestern rodents in association with human disease (19). In the past 10 years, a large number of arenaviruses have been isolated from rodents in southwestern United States. (20,21) and some large cities in the United States harbor rodent populations with as much as 8% seropositivity for arenaviruses (11,22,23). The Old World arenaviruses include Lassa fever virus and several milder species: Mopeia (or Mozambique), Mobala, Ippy, and the prototype laboratory strain, lymphocytic choriomeningitis virus (LCMV). LCMV has worldwide distribution due to the mobility of small mice and the use of this virus in basic laboratory research. Recently, organs from an eastern European donor caused the deaths of three organ recipients in Australia because of the donor’s subclinical infection with an LCMV-like virus (24). Like Lassa virus, the milder African viruses are carried by rodents of the Mastomys species and can infect human beings. However, unlike Lassa, Mopeia from South Africa and Mobala from central Africa have not been known to cause disease and may serve to protect primates. Mopeia-endemic regions of Africa had no reported cases of Lassa-like illness (25) until recently, when five individuals succumbed to a Lassa-like nosocomial infection in South Africa. It is now known that the outbreak was caused by an entirely new arenavirus species, Lujo virus, with 59% and 45% amino acid homology to the nucleocapsid proteins of Lassa and Tamiami viruses respectively (26). In the past two decades, a new arenavirus species has been discovered approximately every three years (27). The evolution of arenaviruses is highly related to coevolution with rodent species: the Old World arenaviruses are carried by Eurasian rodents of the family Muridae and the New World arenaviruses are carried by American rodents in the subfamily Sigmodontinae (28). The absence of Asian arenaviruses is likely due to lack of surveillance in Asia. The relatedness of different species within the arenavirus family can be seen in Figure 1.

Lassa Virus Transmission and Prevalence Lassa virus is the most virulent of the arenaviruses known to cause hemorrhagic fever (29). Nosocomial outbreaks in Nigeria led to its isolation and further characterization in the 1960s and 1970s (30). The most frequent natural host for Lassa virus is a small peridomestic rodent, Mastomys natalensis, which carries Lassa virus as a chronic infection and can transmit it vertically to its offspring and horizontally by shedding (31,32). People are infected by coming in contact with infectious excreta or by ingesting contaminated food. Lassa infection is responsible for significant morbidity and mortality in West Africa, with

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Figure 2 Seroprevalence of Lassa virus in Guinea, West Africa. Studies in 1991 (34) and 2004 (32) give similar data for Lassa virus positive serum IgG found in villagers of the Faranah province of Guinea.

Figure 1 Arenavirus family phylogeny. The Old World arenaviruses include Lassa and other African viruses, as well as the prototype arenavirus, LCMV that is found worldwide. The New World arenaviruses in the Americas are in three subgroups A, B, and C, with hemorrhagic fever being associated with Junin, Machupo, Guanarito, Sabia, Chapare (new), and possibly also White Water Arroyo virus. Abbreviation: LCMV, lymphocytic choriomeningitis virus. Source: From Ref. 16.

approximately 300,000 infections and 3000 to 5000 deaths annually (25,29,33). It is estimated that 10% to 50% of adults in Lassa endemic areas are seropositive, and around 10% to 25% of infected individuals experience acute disease. A study in villages of Guinea showed little change in seroprevalence over a 17-year period (32,34) (Fig. 2). Death occurs in the absence of treatment for 15% to 20% of people with severe, acute disease, and in 2% to 10% of those receiving ribavirin treatment. In 2000, four lethal Lassa fever cases involving travelers from Africa were diagnosed in Germany, United Kingdom, and the Netherlands (35). Deterioration of social and economical conditions in areas of high endemicity, especially Sierra Leone and Liberia, significantly increased the incidence and mortality of this disease (36). Ribavarin is the preferred treatment for Lassa infection (37), but often comes too late. Prevention by means of a vaccine has advantages over treatment with antivirals (36,38).

Lassa Virus Pathogenesis in Different Models Most information on the molecular basis of arenavirus pathogenesis has been obtained from murine infections with LCMV. In the mouse, pathology is associated with a vigorous cytotoxic T lymphocyte response that can be blocked by immunosuppressive agents (39,40). Unlike studies in the mouse (41), arenavirus disease in guinea pigs or primates is not relieved by immunosuppressive agents (42). The virulence of Lassa

virus in man, guinea pigs, and primates is directly related to virus loads (43–45) and is unlike the classical immunopathogenesis in mice. Furthermore, the murine models lack some of the toll-like receptors and gd T-cell responses of primates (46). Strain 13 guinea pigs are sensitive to Lassa virus infection such that 100% of inoculated animals can be expected to succumb to a lethal dose, c.a. 1000 plaque-forming units (pfu), whereas only a third of outbred Hartley guinea pigs are sensitive to Lassa virus infection (47,48). Unfortunately, there are insufficient guinea pig-specific reagents to make that model more useful, so primates continue to be best for Lassa fever studies. Rhesus macaques are considered the best model for human Lassa fever with disease onset occurring approximately seven days after exposure (38,45,49–51). Cynomolgus macaques (52) and marmosets (3–5) take slightly longer to exhibit disease signs but also provide excellent models that mimic Lassa fever in human beings. Lassa and other hemorrhagic viruses such as Ebola are acute viral pathogens (53). Human survivors manage to control their viral loads, whereas fatal cases have high unrelenting viremia (54). The most consistent pathologic finding is hepatocellular necrosis, although the extent of damage is usually insufficient to implicate hepatic failure as the cause of death (29,37,43). Other signs include myocarditis and facial and pulmonary edema, and in severe advanced cases, hemorrhage and hypovolemic shock resulting from vascular leakage (37,43).

Role of Chemokines in Protection Studies in cell culture and in vivo indicate that virulent Lassa virus infection inhibits the cell-signaling pathways and chemokine production that are seen in mild arenavirus infections. When Lassa (virulent) and Mopeia (avirulent) infections were compared in cultured monocytes and endothelial cells, Lassa virus, in contrast to Mopeia virus, did not induce tumor necrosis factor (TNF)-a expression and downregulated IL-8, an inflammatory chemokine that attracts neutrophils to sites of infection (55,56). It is possible that Lassa suppresses neutrophil activation and thereby evades an early host defense resulting in greater virulence for Lassa than for Mopeia. Severe Lassa fever

Chapter 82: Vaccines Against Lassa Fever

is characterized by sustained neutrophilia, supporting the view that neutrophils are activated but not drawn to infected tissue sites. Clinical studies of Lassa fever patients by Mahanty et al. (57) confirmed our in vitro findings and showed no elevation of TNF-a in serum. Low or undetectable levels of IL-8 were correlated with poor outcomes. The Pichinde-infected guinea pig model for Lassa fever was investigated using kinomics and biochemical approaches to show that the virulent infections were remarkable for inhibiting cell-signaling (especially through the NF-kB pathway), whereas mild infections had normal signaling (48,58). Our experiments on nonhuman primates using LCMV-WE-infected monkeys as a model for Lassa fever (59–62) gave further support to these findings. The virulent infection was associated with undetectable levels of TNF-a, low levels of IL-8, and high levels of IL6 in plasma (59,60). Similarly, a microarray analysis of peripheral blood mononuclear cell (PBMC) mRNA from infected monkeys showed that monkeys with a virulent (LCMV-WE) infection suppressed IL-8 mRNA, whereas monkeys with a mild (LCMV-Armstrong) infection did not (63), contributing to the view that mild infection promotes protective innate immune responses.

Molecular Biology of Lassa Virus Like most negative-stand RNA viruses, the Arenaviridae have a small single-stranded RNA genome of approximately 11 kilobases (Fig. 3A, B). The Salvato laboratory completed the first fulllength arenavirus sequence (64) as well as additional sequences of virulent and attenuated strains (65–69). Arenaviruses have two genome segments, each with bidirectional coding

Figure 3 Lassa virus is an enveloped virus with two single-strand RNA genome segments. (A) An electron micrograph of a budding Lassa virion from a cultured cell. The electron micrograph is by I. Lukashevich. (B) Genome structure and antisense coding of Lassa genes reveals the L RNA segment of 7.2 kb encoding the Z protein in the positive sense, and the L protein in the negative sense. The S RNA segment of 3.4 kv encodes the envelope glycoprotein (GP) in the positive sense and the nucleocapsid protein (NP) in the negative sense.

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arrangements and complementary termini (27). The large (L RNA) segment encodes the L protein (or polymerase) on the anti-genomic (negative) sense and a small zinc-binding protein (Z) in the positive sense. The small (S RNA) segment encodes the viral glycoprotein (GP-C) in the positive sense and the NP in the negative sense. The GP undergoes cotranslational processing to form a signal peptide (SP) that mediates the cleavage-maturation of GP-1 and GP-2 from the GP-C precursor (70,71). The small Z protein is a zinc-binding matrix protein that is involved in virus uncoating and budding (64,72,73), inhibiting polymerase activity (74–76), scaffolding for virus assembly (77), and acting as a secretagogue of infected cells (78). Overexpression of Z prevents viral transcription (76) and promotes a persistent infection (79,80). Exchange or reassortment of genome segments has been used to map viral phenotypes (81–83). The small (S RNA) segment encodes activities that contribute to virulence, NP (84), and GP (85). However, the overriding requirement for virulence is encoded on the L RNA where some function of the polymerase and/or Z protein determines the extent of virus replication at infected sites (66,81,83,86). Although NP apparently inhibits signaling and GP determines tropism, the deleterious functions of these structural proteins have no impact on the host without a high level of transcription being driven by genes on the L RNA. One could imagine that high replication could be encoded entirely by the L polymerase protein or by a less tenacious inhibitory function encoded by the Z protein. The vaccine strain Mopeia/Lassa reassortant 29, ML29, has a Mopeia L segment and a Lassa virus S segment. Thus ML29 has the nonvirulent phenotype and plaque-forming morphology of Mopeia, with the structural proteins of Lassa virus (3,4,82,83). Eighteen additional mutations distinguish the ML29 genome from the parental strains and likely contribute to the attenuated phenotype. Reverse genetics for the arenaviruses in general (74,87,88) and Lassa virus in particular (75) are available and will soon resolve issues about the exact determinants of virulence and the optimum sequences for vaccines strains. The genetic diversity of the Lassa virus poses a great challenge for vaccine development. Lassa virus represents a diverse group of viruses with overall strain variation as high as 27% at the nucleotide level (89). Lassa virus isolates comprise four phylogenetic lineages, three of which are found in Nigeria, a fourth in Guinea, Liberia, and Sierra Leone, and recently, a fifth lineage has been proposed, Lassa-AV, from a patient with initials AV infected in Ghana or Ivory Coast (35). The NP is the most variable gene and encodes structural proteins (89) with the highest sequence differences, based on partial NP sequences, between lineages II (803213/NIG/74/H) and IV (JOS/ SL/76/H), 11.0% to 14.4%. The attenuated vaccine candidate ML29 has so far been the only candidate successful in crossprotective challenge experiments against the most divergent Lassa strains (4).

LASSA VACCINE STRATEGIES Why a Lassa Vaccine Is Needed? Programmatic use of a vaccine in endemic areas could reduce the incidence of Lassa fever. A vaccine would also be useful for protecting laboratory workers who work with Lassa virus and for health care workers who care for patients with Lassa fever in outbreaks and endemic situations. It is reasonable to expect that different vaccination strategies would be applied to

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different vaccine recipients. In endemic areas, where most of the target population is poor and many live far from health care facilities, a single-dose vaccine would be ideal. A multidose immunization regimen might be practical for medical staff working in endemic areas. Once there is an outbreak, a fastacting vaccine or postexposure prophylaxis would be best. Lassa fever could be controlled by reducing the rodent population around human habitats or by limiting their access to human beings. However, this is impractical in the endemic areas of rural West Africa, especially since rodents aggregate wherever humans store their food; moreover, in some areas, the rodents themselves are sometimes used as a food source (90). In theory, a vaccine could practically be targeted to the rodents as well as to humans. Rabies vaccines in the form of meat baits laced with attenuated rabies virus are routinely airdropped into endemic areas of the United States by the U.S. Department of Agriculture, effectively curbing the incidence of rabies carried by wild raccoons, rodents, foxes, and wolves (91). By diminishing the sylvatic animal reservoir of rabies, risk to humans and their domestic animals is diminished. Theoretically, a similar strategy could be used in the endemic areas of West Africa in which attenuated vaccines could be sprinkled on food bait and left for rodents to eat. Because of the many hurdles of developing a vaccine for humans (huge costs, high standards of safety and efficacy, etc.,) vaccinating the animal reservoir rather than the human population has attractions.

Nonreplicating Vaccines Inactivated Virus Particles Conflicting reports have been published: one claiming success and the other failure of inactivated Lassa vaccines. The successful group used a single dose of b-propiolactone-inactivated Lassa virus and protected half of the vaccinated Papio hamadryas monkeys from disease and viremia after an intramuscular challenge with a 0.4 pfu dose of Lassa virus (92). Another group used g-irradiated Lassa virus. Despite stimulating humoral responses to the NP and GP viral proteins, they saw no protection in all three of the three challenged rhesus macaques (93). It may be that the different monkey models, inactivation methods, and the lower challenge dose in the first model account for the different results. DNA Vaccines Whitton and colleagues explored the efficacy of DNA vaccination (94) and were able to achieve 50% protection of vaccinated mice challenged by the intracerebral route with LCMV. This murine challenge model is far from the primate disease, but since it requires a good cell-mediated immune response to prevent death, the model provides a quick and inexpensive test of a vaccine’s ability to elicit cell-mediated immunity (CMI). This group (95) went on to test DNA vaccination with a plasmid encoding Lassa NP, and showed they could protect 50% of mice from a heterologous challenge with LCMV or Pichinde viruses. They also showed that a minigene expressing a Lassa NP epitope conferred protection, thus suggesting that CMI mediated protection. This line of investigation was not pursued in guinea pigs or primates. Schmaljohn and coworkers gave guinea pigs and cynomolgus macaques multiple gene gun inoculations with plasmids expressing filovirus immunogens, and based on their data, concluded that DNA vaccination had borderline efficacy for primates (96).

Peptides, and Nonreplicating Alphavirus Vector Vaccines Using computer-assisted algorithms, five HLA-A2.1-binding Lassa virus GP peptides and two NP peptides were identified (97,98). HLA-A*0201 transgenic mice immunized with either Lassa virus peptide GP-C(42–50) or GP-C(60–68) were protected against challenge with a recombinant vaccinia virus that expressed Lassa GP. However, peptide-based vaccination may have pathological consequences in individuals recently infected with the virus or in immune individuals previously exposed, perhaps unknowingly, to the pathogen (99). This is exactly the case for Lassa virus infection in West Africa where 30% of all infections are asymptomatic and seroprevalence can be as high as 25% to 55% (34,51,90). Other healthy Lassa virus– exposed individuals in endemic areas have been described who lack antibodies but manifest strong proliferative responses to Lassa NP and GP-2 proteins (100). Particles containing Lassa viral antigens can also serve as Lassa vaccines. Pushko et al. (101) used an attenuated Venezuelan encephalitis virus (VEE) replicon system to express Lassa NP engineered to replace the structural proteins of VEE. The VEE capsid and envelope proteins were supplied in trans to package the NP into VEE replicon particles (VRP). These particles elicited antibody against Lassa NP in Balb C mice. In later studies, VRP expressing either NP or GP of Lassa virus successfully protected strain 13 guinea pigs from Lassa virus challenge (8). Guinea pigs had each been given three subcutaneous vaccinations (with 107 particles of VRP-NP, VRP-GP, or a mixture of GP and NP) at four-week intervals and then challenged with 103 pfu of Lassa virus. Whereas all of the vaccinated animals were free of clinical disease, several developed viremia by day 7 postchallenge including three of the four animals given VRP-GP, one of four animals given VRP-NP, and one of four animals given both NP and GP. All animals developed antibodies, although none were neutralizing; CMI was not measured. Thus, clinical protection without sterilizing immunity could be achieved by VRP immunization of guinea pigs. At present, Lassa antigen production via VRP is not costeffective and the need for multiple inoculations is not a practical strategy for vaccinating the African populations. Discovery of a more cost-effective way to produce Lassa subunit particles would make this a more attractive approach. In 2006, Urata et al. (102) reported that cellular protein TSG101 was critical for virus budding, and that the Lassa Z protein could mediate budding, so there is potential for harnessing Lassa viral proteins to form virus-like particles.

Replicating Vaccines The Case for Replicating Vaccines A replication-competent ‘‘live’’ vaccine is an attractive approach for controlling Lassa fever because it can provide (i) the most effective natural pathway for processing and presenting viral molecules to the host, (ii) long-lasting protection after a single dose (51), and (iii) if formulated to provide both GP and NP antigens, it can be broadly cross-protective (3–5). Epidemiological studies of natural infections indicate that strong CD4+ memory T cells against many Lassa strains can persist for 15 years (100,103). Early Studies and Cell-Mediated Immunity Early Lassa vaccine studies that used vaccinia live vectors showed that both GP and NP are effective immunogens in guinea pigs (104,105). However, in nonhuman primates, immunizations

Chapter 82: Vaccines Against Lassa Fever

with NP, GP-1, or GP-2 alone were ineffectual, while the full precursor glycoprotein (GP-C) could be effective (38). Circumstantial evidence indicated that CMI was required for protection from lethal Lassa virus challenge. Lassa survivors, convalescing primates, and guinea pigs often lack neutralizing antibody responses (38,47,106). First, neutralizing antibodies often appear late in infection, and the transfer of ‘‘immune’’ plasma is rarely effective for protection against disease in animal or human trials (37,107). Second, g-irradiated preparations of purified Lassa virions elicited antibodies in monkeys to all three major structural proteins, NP, GP-1, and GP-2, but did not protect against Lassa challenge (93). Third, live virus vaccines and live vector vaccines that elicit CMI (e.g., Lassa-expressing vaccinia recombinants, Salmonella recombinants, or Mopeia) elicited protective immunity in animals (38,49,108–110). Fourth, adoptive transfer of spleen cells protects against Lassa challenge in a mouse model (44,83). Finally, it has been demonstrated that Lassa-seropositive persons have strong CD4+ T-cell responses against Lassa NP (100). Thus, CMI is likely to be essential to survive Lassa virus infection. Humoral responses play a minor role in protection from arenaviral hemorrhagic fevers (44,51). Neutralizing plasma from monkeys with titers above 10,000 could protect Lassa-infected cynomolgus macaques, and were especially effective when given with ribavirin (52,107). Convalescent plasma from Lassa patients has also protected guinea pigs when the titer of neutralizing antibodies exceeds 100; however, this level of antibody is difficult to obtain from human plasma (111). In experimental primate infections anti-viral serum titers are often attended by high antigen-driven cell-mediated responses, but these are rarely correlated with good neutralizing titers. Thus, in assessing Lassa vaccines, it is important to measure cell-mediated responses. Identifying Protective Antigens Reassortant analysis indicates that the S RNA of LCMV and Lassa viruses encode the major antigens eliciting protective immunity in mice and guinea pigs (83,112), and detailed studies in mice have identified the precise epitopes involved. LCMV NP expresses the immunodominant CTL epitope (NP 118–126 aa) for H-2d mice (113). Within that epitope, a minimal tetrapeptide GVYM is identical for LCMV, Lassa, and Mopeia viruses and plays an important role in CTL recognition (114). The LCMV NP 118–126 epitope shares 78% homology with Lassa and Mopeia NP, and constitutes more than 97% of the total bulk CTL produced in BALB mice. A peptide consisting of amino acid residues 118–126 derived from the NP of LCMV, Lassa and Mopeia viruses binds at high affinity to MHC molecule Ld (115). Recombinant Lassa NP protein expressed in vaccinia or in Salmonella protects a third of LCMV-challenged mice, and protection is associated with cross-reactive CTL and proliferative responses (108,109). Similarly, cross-protection against LCMV challenge has been induced by Lassa GP-2-derived peptide, residues 403–417, corresponding to a highly homologous sequence on the glycoprotein gene of Lassa and LCMV (116). Finally, it has been shown that cross-reactive GP-2derived epitopes are involved in human CD4+ T lymphocyte responses against Lassa virus (103). Early studies of Auperin, Fisher-Hoch, and McCormick that include data from 44 nonhuman primates given recombinant vaccinia expressing Lassa NP, GP-C, GP-1, GP-2, and combinations of these proteins were reviewed. All animals given vaccinia expressing single glycoproteins GP-1 or GP-2 died (38,50,51,117). However, vaccinia virus expressing the

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Lassa virus precursor glycoprotein (GP-C) protected eight of nine primates from Lassa fever after a single subcutaneous (SC) inoculation, and seven of eight primates were protected by vaccinia expressing both Lassa NP and GP-C. Only 3 of 11 monkeys given vaccinia expressing NP were protected. Fisher-Hoch and McCormick concluded that the full glycoprotein (GP-C) is necessary and sufficient to protect primates from lethal challenge; however, since animals vaccinated with NP alone faired poorly, they suggested that larger studies were needed to determine whether NP was actually potentiating viremia following challenge (50). Importantly, vaccination protected monkeys from lethal disease, but not from infection, and viremia in animals vaccinated with GP-C + NP was three logs lower than in GP-C-vaccinated monkeys. Thus, NP may be needed in a vaccine to achieve sterilizing immunity. Recent studies (118) showed that CTL against early LCMV NP-derived epitopes are more important in virus control than those against late GP-derived epitopes. Mice that are tolerant to all NP-derived T-cell epitopes were severely compromised in their ability to control larger inocula of LCMV, supporting the hypothesis that CD8 T cells specific for early viral antigens play a major role in acute virus control. Recombinant Lassa NP protein protected all guinea pigs from subsequent Lassa virus challenge (104), whereas, in the primate model only 20% of NP-vaccinated macaques were protected (38). On the other hand, Lassa virus-infected individuals from Guinea had strong memory CD4 responses against the NP, which were partly strain-specific and partly cross-reactive with other Lassa virus strains (100). Recent cell culture studies implicate NP in blocking type I interferon responses (84). However, this anti-interferon activity was found in both pathogenic and nonpathogenic arenaviruses. The NP protein of Tacaribe virus, the only arenavirus isolated from fruit bats, had no anti-interferon activity. It is possible that the anti-interferon phenotype is specific for rodent-carried arenaviruses, and may be associated with establishment and/ or maintenance of persistent infection in rodents rather than with pathogenicity of arenaviruses in human beings. Details About the ML29 Vaccine The ML29 vaccine is a highly promising vaccine candidate, as evidenced by details of a recent study (5) summarized below. Six marmosets were vaccinated with ML29 and challenged a month later with 103 pfu of Lassa-Josiah. By day 8 to 10 the four unvaccinated control animals became depressed, anorexic, and lost 10% of their body weights. The disease gradually progressed, and on day 17 to 21 all controls met anesthesia criteria. The vaccinated animals had no clinical manifestations, no remarkable blood chemistries or hematology, and survived the 35-day observation period. In contrast, controls showed reduced platelet numbers, elevated liver enzymes, and decreased plasma albumin. Lassa virus was detectable in control marmosets by day 5 after challenge, with more than three logs of viremia at necropsy. One ML29-vaccinated marmoset had detectable virus by plaque assay on day 5, and two others had virus detectable by a more sensitive cocultivation assay, but by day 15 after Lassa challenge, there was no detectable virus by any assay, including Reverse TranscriptionPolymerase Chain Reaction (RT-PCR). Although a 103 pfu dose of ML29 vaccine was sufficient for protection, eight marmosets were given a high dose of 106 pfu to determine whether there were any unforeseen toxicities at high dose. Most of the low-dose marmosets had

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America it is regarded as a risk group 2 pathogen, and can be manufactured in conventional facilities. For example, production of the attenuated arenavirus vaccine for the agent of Argentine hemorrhagic fever, Junin virus, takes place outside of BSL-3 in Argentina (119).

SUMMARY OF VACCINE RESULTS

Figure 4 Survival of ML29 vaccinated marmosets after lethal challenge with Lassa virus. Six animals were vaccinated SC with ML29. Four control animals were mock-vaccinated with saline. On day 30, all were challenged with Lassa virus. Vaccinated animals had no clinical signs of disease and were necropsied at the end of a 35-day observation period.

cleared the vaccine strain by day 28 after vaccination, but residual virus could be detected in some animals. No histopathology was observed in an extensive examination of tissues from vaccinated and Lassa virus–challenged animals. TNF-a ELISPOT and IgG ELISA showed robust immune responses by day 21 after ML29 vaccination. In the end, six of six vaccinated animals were protected, and four of four unvaccinated controls succumbed to lethal challenge (Fig. 4). GMP production of ML29 is encumbered in the United States by its classification as a risk group 3 pathogen. However, in Europe and South

A comparison of Lassa vaccination studies (Table 1) in animal models allows us to make the following conclusions: (i) Mopeia is an excellent natural vaccine; (ii) inactivated Lassa virus protects poorly or not at all (92,93); (iii) vaccinia vectors expressing the full Lassa glycoprotein protect well, (38,105), as do alphavirus, vesicular stomatitis virus (VSV) and yellow fever (YF) vectors expressing GP (6–8); and (iv) DNA vaccines expressing Lassa NP (95) and a Salmonella vector expressing NP (108,109) generate cell-mediated responses in mice, but it is not known if they can offer sufficient protection for primates. Although vaccinia vectors expressing Lassa immunogens have protected, the vaccinia vector may not be sufficiently safe for immune-suppressed individuals (50). The DNA and alphavirus vaccines are not practical as stand-alone vaccines because they would require multiple inoculations. The Salmonella vector failed to work as a single-dose immunogen in primates (Salvato, unpublished). So far, vectors expressing GP alone (e.g., VSV-LAS and YF-LAS) have not registered CMI prior to challenge and have not been entirely cross-protective for heterologous challenge viruses (6,7). Furthermore, the VSV vector has issues with neurotropism (120). The ML29 is potentially cheap to produce, elicits strong immune responses before challenge, confers broad protection, and is not neurotropic. Moreover, unlike any of the other candidate vaccines, it resulted in sterilizing immunity in the guinea pig and monkey models (3–5). Previous studies established that the GP-C immunogen confers protection, and raised the possibility that NP immunogens diminish or promote viremia and may be critical for cross-protective immunity. Currently, we are testing the safety

Table 1 Efficacy of Various Lassa Candidate Vaccines or Hyperimmune Sera in Preventing Lassa Clinical Disease or Death in Different Animal Models Challenged with Wild-Type Lassa Virus Lassa vaccine or hyperimmune sera Passive antibody Mopeia g-Irradiation-inactivated Lassa virus b-Propriolactone-inactivated Lassa virus Vaccinia (VV-NP) Reassortant Mopeia/Lassa Salmonella-(Lassa) NP Vaccinia VV-GP-1, VV-GP-2 VV-GPc, VV-NP VV-N, VV-G, summary a-Vectored-Lassa VSV-Lassa GP Plasmid encoding Lassa NP Reassortant MOP/LAS YF-LAS

Description

References

Guinea pig or primate sera having high Lassa-neutralizing titers >100 protected strain 13 guinea pigs Protected four of four monkeys Antibody responses but no protection in three macaques

111 37 93

Protected 50% of papio hamadryas monkeys

92

Protects guinea pigs but failed to protect macaques Protected all mice from ic challenge with Lassa Protected a third of mice from ic heterologous challenge with LCMV Both GP-1 and GP-2 needed for full protection of macaques

104 83 108, 109 37, 49, 105, 110

Lassa GPc is necessary and sufficient. NP protects macaques poorly Abortive-VEE replicons delivered Lassa NP or GP to protect strain 13 guinea pigs Macaques protected by im VSV-Lassa GP, no CMI detected before challenge Protected mice from LCMV or Pichinde ic challenge 10 of 10 guinea pigs protected. Also protects from heterologous challenge and challenge on day of vaccination 4 of 5 strain 13 guinea pigs protected from heterologous Lassa challenge

50, 51 8 6 171 3, 4, 5 7

Abbreviations: VV, vaccinia virus; NP, nucleocapsid protein; GP, glycoprotein; VSV, vesicular stomatitis virus; YF, yellow fever; LCMV, lymphocytic choriomeningitis virus; VEE, Venezuelan encephalitis virus; MOP/LAS, Mopeia/Lassa reassortant virus; YF-LAS, Yellow fever - Lassa virus recombinant.

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of the ML29 vaccine in nonhuman primates with simian AIDS. So far, there is no remarkable increase in pathogenicity, as was predicted by Centers for Disease Control (CDC) reports about immune-suppressed transplant recipients succumbing to LCMV-contaminated organs (121). From what we know of the transcriptome profiles of mild arenavirus infections, there is even the possibility that SIV-infected monkeys will exceed their normal lifespans as a result of ML29 infections. One issue we had not anticipated was the preference by some for a yellow fever-vectored vaccine over the ML29 vaccine due to production issues. Despite the fact that YF-LAS has weaker immunogenicity and offers less cross-protection than ML29, the ability to produce it in egg cultures in facilities that are already in place in some developing countries makes YF-LAS attractive. Thus, we are also going forward with efforts to produce YF-NPLas, to test with YF-GPLas, and to determine the effects of preexisting immunity on response to vaccination and on new immune responses to yellow fever. The best scenario would be that the addition of the NP immunogen will broaden the vaccine’s efficacy against Lassa fever without reducing its efficacy against yellow fever.

POSTEXPOSURE VACCINATION Several vaccines are known to be effective after exposure to a lethal dose. Smallpox can be prevented by vaccinia inoculation up to five days after exposure and Varicella zoster can be prevented up to three days postexposure (122). Furthermore, DNA vaccination has been shown to break tolerance to ‘‘infection’’ by prion particles and to cancers (123,124), thus providing a treatment that is in the realm of postexposure vaccinations. Postexposure treatment of Lassa fever by the VSV-LAS vaccine has been reported at meetings but not yet published (125). The ML29 vaccine has also been inoculated at the same time as the Lassa challenge and protected four of four guinea pigs (4). This rapid-protection phenomenon resembles the protection seen when nonhuman primates are inoculated with extremely high doses of Lassa virus. In 2002, Sylvan Baize reported that cynomolgus macaques given 107 pfu of Lassa virus survived whereas most animals given a lower dose, only 103 pfu died (126). The most likely mechanism for this protection is that the high dose elicits a potent protective innate immune response. It may be that an early upregulation of IL-8 brings a polymorphonuclear leukocyte response that destroys virus-infected cells and eliminates the infection before it has a chance to become established.

PROSPECTS FOR THE FUTURE The future promises new methods for efficacy and safety testing and new surrogate markers for immunity. For a promising Lassa vaccine to go forward, one needs validated surrogate markers for protective efficacy, and CMI is high on the list. In nonhuman primate models, the VSV-LAS vaccine failed to display any CMI prior to lethal challenge, thereby leaving the investigators with few criteria for efficacy testing. The ML29 vaccine had detectable CMI within seven days of a single vaccine inoculation, and robust CMI by day 21 after a single vaccine dose. Nevertheless, it is clear that ELISPOT measures are not an adequate surrogate for protective efficacy since animals with no detectable CMI were still protected. Therefore, we have been actively profiling monkeys to detect changes in the transcriptome (or other omics system) that could serve as

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correlates of protection. In a preliminary study, we compared a virulent arenavirus infection to infection with a mild vaccine strain and clearly showed that host responses to the vaccine strain differed from responses to the virulent strain by day 2 to 3 after vaccination (63). For example, the vaccine strain induced high levels of IL-8 mRNA in PBMC by day 3 whereas the virulent strain suppressed IL-8 mRNA. Attempts are underway to validate this and other surrogates of protection in vaccinated nonhuman primates. On a less ambitious scale, transcriptome profiling of the vaccine strain in PBMC cultures is being used as a way to characterize the vaccine inoculum (5).

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83 Hantavirus Vaccines Christopher D. Hammerbeck and Jay W. Hooper United States Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland, U.S.A.

INTRODUCTION Hantaviruses represent a diverse group of rodent-borne viruses within a separate genus of the family Bunyaviridae that cause hemorrhagic fever in humans. Like all members of this family, hantaviruses have a tri-segmented, negative-sense, singlestrand RNA genome that encodes three structural proteins (1,2): the small (S) RNA segment encoding the nucleocapsid (N) protein, the medium (M) segment encoding the envelope glycoproteins Gn and Gc (formerly G1 and G2, respectively), and the large (L) segment encoding the viral RNA-dependent RNA polymerase (RdRp). Hantaviruses are carried by infected rodents and transmitted to humans exposed to contaminated rodent feces, urine, or saliva. The most common mode of transmission is thought to be the inhalation of aerosolized rodent droppings; however, contact with open wounds, rodent bites, and ingestion of contaminated material are also possible modes of transmission. Unlike most hantaviruses, however, there is evidence that the disease caused by Andes virus (ANDV) in Chile and Argentina can spread from person–to person (3–6), illustrating the potential for dissemination of the disease from a single person throughout a large population. Hantaviruses appear to be uniquely linked to their rodent reservoir, such that for any given hantavirus species, there is only one known rodent host (Table 1). Still, while the geographical distribution of hantaviruses that cause disease appears to be distinct, the geographical distributions of rodents known to harbor hantaviruses overlap (Table 1), suggesting that multiple hantaviruses could inhabit a given area. This would increase the risk of potential infection and theoretically could result in the generation of interspecies reassortants. Since over 20 hantaviruses have now been identified, and hantavirus-associated disease has been reported on nearly every continent (Table 2), hantaviruses represent a worldwide health problem. Hantavirus-associated disease frequently occurs in rural areas and correlates with increased numbers of the rodent host, which can be affected by climate and the availability of food (11). Consistent with this, epidemiologic studies report increased incidence of hantavirus disease in persons working or sleeping in environments inhabited by rodents, which include agricultural workers, forest workers, and soldiers (12–15), suggesting that hantavirus disease poses a significant occupational hazard for people living in infectionprone areas. Old World hantaviruses (Table 1) such as Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), and Puumala virus (PUUV) are most common in Europe and Eastern Asia and cause a vascular leak syndrome that primarily

affects the kidneys [hemorrhagic fever with renal syndrome HFRS)]. New hantavirus species including Sin Nombre virus (SNV) and ANDV identified in the Western Hemisphere (Table 1) have been linked to an acute vascular leak syndrome that primarily affects the lungs [hantavirus pulmonary syndrome (HPS), also known as hantavirus cardiopulmonary syndrome (HCPS)] (16,17). Whereas approximately 10% to 15% of HFRS cases are clinically severe, with 6% to 15% of cases becoming fatal, fatality rates for the most common HPS hantaviruses range from 30% to 50% (18). Treatment of patients with HPS or HFRS remains largely supportive and may involve dialysis in severe cases of HFRS or require venoarterial extracorporeal membrane oxygenation (ECMO) therapy to provide cardiopulmonary support during severe cases of HPS (reviewed in Refs. 10,19,20). The only drug to show any clinical efficacy against HFRS, ribavirin, has been shown to reduce viral titers, increase survival rates, and reduce the severity of HFRS in patients in China (21), if administered early after the first symptoms of disease. In contrast, the use of ribavirin as a treatment for HPS does not appear to be as successful (22,23). Ribavirin is not licensed in the United States by the Food and Drug Administration (FDA) for use as a treatment for HFRS, although it has been used off-label after potential exposures to hantaviruses. Somewhat surprisingly, given the worldwide distribution of hantaviruses, only a few HFRS vaccines have been licensed (mainly by regulatory agencies in Asian countries), and there are no vaccines to prevent HPS. Still, an ongoing effort to develop vaccines for these viruses has resulted in many types of experimental vaccines (Table 3). In particular, efforts to develop safe and effective molecular subunit vaccines against multiple hantaviruses have shown promise in animal models. Here, we review the ongoing efforts to develop vaccines against these dangerous pathogens.

VACCINES Inactivated Virus Vaccines Live-attenuated hantavirus vaccines have not been produced. In contrast, inactivated viruses have been used to generate vaccines based on HTNV, SEOV, and PUUV (reviewed in Refs. 64,65) that have been licensed for use in South Korea and China. In South Korea, Hantavax 1 was created and licensed based on the observation that high viral titers can be obtained from rodent brains inoculated with hantavirus (27). The process of manufacturing Hantavax involves formalin inactivating HTNV derived from infected mouse brains,

BAYV ARAV CASV CHOV

Hu39694 JUQV

Bayou virus Araraquara virusc Castelo dos Sonhos virusc Choclo virusc

Human 39694 virusc Juquitiba virusc

Bayou virus

Unknown Unknown Unknown

Unknown Unknown

SNV

Sin Nombre virus LNV NYV BCCV

MGLV

BMJV LECV MCLV ORNV

Bermejo virus Lechiguanas virus Maciel virus Ora´n virus Monongahela virus

ANDV

Andes virus

PUUV

Laguna Negra virus Laguna Negra virus New York virus New York virus-RI-1 virus Black Creek Canal Black Creek Canal virus virus

Sin Nombre virus

Andes virus

K27 virusc Sotkamo virus

SAAV

Saaremaa virus

Puumala virus

DOBV

Dobrava virus

Dobrava–Belgrade virus

SEOV

HTNV

Hantaan virus

Seoul virus

AMRV

Amur virus

Abbreviation

Seoul virus

Hantaan virus

Representative virus strain

Unknown Oligoryzomys nigripes (black-footed pygmy rice rat)

Bolomys lasiurus (hairy-tailed bolo mouse) Unknown Oligoryzomys fulvescens (fulvous pygmy rice rat)

Oryzomys palustris (marsh rice rat)

Calomys laucha (Small vesper mouse) Peromyscus leucopus (white-footed mouse) Sigmodon hispidus (hispid cotton rat)

Peromyscus maniculatus (deer mouse)

Oligoryzomys longicaudatus (long-tailed pygmy rice rat) Oligoryzomys chacoensis (Chacoan pygmy rice rat) Oligoryzomys flavescens (yellow pygmy rice rat) Necromys benefactus (Argentine bolo mouse) Oligoryzomys longicaudatus (long-tailed pygmy rice rat) Peromyscus maniculatus (deer mouse)

Clethrionomys glareolus (bank vole) Clethrionomys rufocanus (gray red–backed vole)

Apodemus flavicollis (yellow-necked mouse) Apodemus agrarius (striped field mouse)

Rattus losea (lesser rice-field rat) Apodemus agrarius (striped field mouse)

Rattus norvegicus (brown/common/Hanover/ Norway/Norwegian or wharf rat) Rattus rattus (house/black or roof rat)

Apodemus agrarius (striped field mouse)

Apodemus peninsulae (Korean field mouse)

Rodent reservoir

c

b

Hemorrhagic fever with renal syndrome. Hantavirus pulmonary syndrome. Virus not listed in Virus Taxonomy: The Eight Report of the International Committee on Taxonomy of Viruses. Source: From Refs. 7–10.

a

HPSb

HFRSa

Disease Virus species

Table 1 Old and New World Hantaviruses Known to Cause Human Disease

Argentina, Brazil, Paraguay

Belize, Brazil, Colombia, Costa Rica, Ecuador, El Salvador, French Guiana, Guatemala, Guyana, Honduras, Mexico, Nicaragua, Panama, Suriname, Trinidad and Tobago, Venezuela

Alaska and Canada, western and central United States, central Mexico Alaska and Canada, western and central United States, central Mexico Argentina, Bolivia, Brazil, Paraguay, Uruguay Northeast through southwest United States, Mexico Southeastern through southcentral United States, Mexico, Central America through northern South America Southeastern through southcentral United States and along the Atlantic coastal plain Argentina, Bolivia, Brazil, Paraguay

Argentina, Bolivia, Brazil, Paraguay Argentina, Brazil, Uruguay Argentina Argentina, Chile

Argentina, Chile

All continents except Antarctica; most common in coastal areas and tropical regions China, Laos, Taiwan, Thailand, Vietnam Central and Eastern Europe, Central Asia, southern Siberia, Manchuria, Korean Peninsula, southeastern China, Taiwan Southern Europe through Scandinavia, Britain Central and Eastern Europe, Central Asia, southern Siberia, Manchuria, Korean Peninsula, southeastern China, Taiwan Western and Central Europe Central Europe through Scandinavia, far east Russia, northern China, Korean Peninsula

Far east Russia, northern China, Korean Peninsula, northern Japan Central and Eastern Europe, Central Asia, southern Siberia, Manchuria, Korean Peninsula, southeastern China, Taiwan All continents except Antarctica

Geographical range of rodent reservoir

906 Hammerbeck and Hooper

Chapter 83: Hantavirus Vaccines

907

Table 2 Worldwide Distribution of Hantavirus Disease Disease

Country

Number of cases (years)

Major hantavirus species

20,000–50,000 (per year) 316 (per year) 100–400 (per year)

HTNV, SEOV

3000 (per year)

HTNV, SEOV PUUV

372 (2005b) 487 (1952–1995) 399 (1952–1995) 134 (1952–1995) 10 (per year) 1000 (per year) 253 (2005b) 448 (2005b) 21 (1952–1995) 136 (1952–1980) 14 (1984–1987) 14 (2005b) 10 (1952–1995) 129 (1952–1995) 27 (2005b) 50 (per year) 228 (1952–1995) 10 (per year) 106 (1952–1995) 300 (per year)

DOBV, PUUV

88 (1993–2004) 465 (1993–2007)

BAYV, NYV, BCCV, SNV

35 (1993–2004)

CHOV

592 (1993–2004)

ANDV, ARAV, CASV, Hu39694, JUQV

HFRS Eastern Asia/Pacific rim China North Korea South Korea Australia, Fiji, Hong Kong, India, Indonesia, Japan, Malaysia, Mongolia, Myanmar, Singapore, Sri Lanka, Taiwan, Thailand, and Vietnama Eastern Europe/Russia Russia (eastern) Russia (western) Belarus, Estonia, Georgia, Latvia, Lithuania, Poland, Romania, and Ukrainea Western Europe/Scandinavia Belgium Bosnia-Herzegovina Bulgaria Croatia Denmark Finland France Germany Greece Hungary Italy Luxembourg Macedonia Montenegro Netherlands Norway Serbia Slovakia Slovenia Sweden Albania, Austria, Czech Republic, Great Britain, Portugal, Switzerlanda HPS North America Canada United States Central America Panama South America Argentina Bolivia Brazil Chile Columbia Paraguay Uruguay Venezuela

36 (1993–2004) 423 (1993–2004) 331 (1993–2004) 8 (2006) 99 (1993–2004) 48 (1993–2004) 2 (1993–2004)

a

Countries reporting rare or sporadic cases of hantavirus disease, or seroepidemiologic evidence of hantaviruse infection. Epidemic year.

b

followed by precipitating, purifying and combining the inactivated virus with aluminum hydroxide as an adjuvant. Hantavax has been shown to protect mice against infection (24,25,27–29,64,65). Similarly, both Hantavax and a related bivalent vaccine (HTNV and PUUV) produced in hamster brains are able to induce seroconversion and elicit neutralizing antibodies in humans, as measured by immunofluorescence and, to a lesser extent, by plaque reduction neutralization.

While Hantavax has been well tolerated in clinical experience in humans without any serious adverse events, less than 50% of the vaccinated population exhibited neutralizing antibodies after 12 months. An epidemiologic study on the efficacy of Hantavax has suggested that the protective effects in South Korea could merely be due to chance (66). A similar cell culture–derived vaccine that has shown complete protection in mice has been found to elicit greater neutralizing antibody

PUUV

Virus-like particle/ chimeric viruses

Recombinant protein

DNA vaccines

Virus-like particle/ chimeric viruses

HBV chimera–expressed PUUV Np peptides þ adjuvant HBV chimera–expressed PUUV Np peptides þ/ adjuvant Polyoma chimera–expressed Np peptides þ adjuvant

Not tested Not tested

Mouse

No No Most Yes Yes Yesa Yes No Yesa Yesa Yesa Somea Not tested Not tested Yes Yes Yes Most Not tested Yes Some

Hamster Hamster Hamster Hamster Hamster Rhesus Hamster Hamster Rhesus Rhesus Rhesus Mouse Mouse Mouse Bank vole Bank vole Bank vole Bank vole Mouse Mouse Bank vole

Mouse

Not tested

Mouse

Not tested

Not tested

Mouse

Mouse

Yes

Yes Most Some Yes Not tested

Yes Some Mosta Somea Mosta Some Mosta

Somea

Protection

Mouse

Hamster Hamster Hamster Hamster Mouse

Mouse Mouse Human Human Human Gerbil Human

Formaldehyde-inactivated Vero E6 cells Formaldehyde-inactivated mouse brain Formaldehyde-inactivated GHKC cells Formaldehyde-inactivated MUKC cells b-Propiolactone inactivated MGKC cells b-Propiolactone inactivated MGKC cells b-Propiolactone inactivated MGKC cells (HTNV þ SEOV) Baculovirus-derived HTNV Np Baculovirus-derived HTNV Gn Baculovirus-derived HTNV Gc Baculovirus-derived HTNV Gn/Gc E. coli-derived HTNV NP fused/complexed with hHSP70 VSV pseudotype expressed HTNV Gn/Gc þ adjuvant HBV chimera expressed HTNV Np peptides þ adjuvant HBV chimera expressed HTNV Np peptides þ/ adjuvant Recombinant vaccinia expressed HTNV Np Recombinant vaccinia expressed HTNV Gn Recombinant vaccinia expressed HTNV Gc Recombinant vaccinia expressed HTNV Gn/Gc Recombinant vaccinia expressed HTNV Np/Gn/Gc Recombinant vaccinia expressed HTNV Np/Gn/Gc DNA vaccine Gn/Gc DNA vaccine Gn/Gc (HTNV þ ANDV) DNA vaccine M gene DNA vaccine M gene (HTNV) DNA vaccine M gene (HTNV þ ANDV) DNA vaccine Gc—hIL-2 fusion gene DNA vaccine S gene—hHSP70 gene fusion DNA vaccine chimeric S–M gene fragments Baculovirus-derived PUUV Np þ adjuvant E. coli–derived PUUV Np þ adjuvant E. coli–derived PUUV Np þ adjuvant Yeast-derived PUUV Np þ adjuvant Plant-derived PUUV Np E. coli–derived PUUV Np linked to K. pneumoniae rP40 HBV chimera–expressed Np peptides þ adjuvant

Recombinant protein

Human

Formaldehyde-inactivated mouse brain (Hantavax)

Inactivated virus

HTNV

Animal model

Source of immunogen

Type of vaccine

Virus targeted

Table 3 Vaccines Against HFRS- and HPS-Associated Hantaviruses

tested tested tested tested tested

tested tested tested tested tested tested tested

tested tested tested tested

Not tested

HTNV, PUUV, DOBV

HTNV, PUUV, DOBV

Not Not Not Not

Not tested Not tested Not tested Not tested No (SEOV, PUUV) DOBV HTNV, SEOV, DOBV No DOBV HTNV, SEOV, DOBV HTNV, ANDV, BCCV Not tested Not tested Not tested Not tested Not tested

HTNV, PUUV, DOBV

HTNV, PUUV, DOBV

Not tested

Not Not Not Not Not

Not Not Not Not Not Not Not

Not tested

Cross-reactivity

(31) (31) (31) (31)

Gedvilaite et al., 2004 (49)

Geldmacher et al., 2005 (36)

Geldmacher et al., 2004 (34,35)

Schmaljohn et al., 1990 (31) Schmaljohn et al., 1990 (31) Schmaljohn et al., 1990 (31) Schmaljohn et al., 1990 (31) Chu et al., 1995 (37) Hooper et al., 2001 (38) Hooper et al., 2001 (38) Hooper et al., 2006 (39) Hooper et al., 2001 (38) Hooper et al., 2006 (39) Hooper et al., 2006 (39) Hao et al., 2008 (40) Li et al., 2008 (32) Zhang et al., 2007 (41) Lunkvist et al., 1996 (42) Lunkvist et al., 1996 (42) De Carvalho et al., 2002 (43) Dargeviciute et al., 2002 (44) Khattak et al., 2004 (45) Maes et al., 2008 (46) Ulrich et al., 1998, 1999 (47,48)

Geldmacher et al., 2005 (36)

Geldmacher et al., 2004 (34,35)

Lee et al., 2006 (33)

Schmaljon et al., 1990 Schmaljon et al., 1990 Schmaljon et al., 1990 Schmaljon et al., 1990 Li et al., 2008 (32)

Sohn et al., 2001 (24); Cho and Howard, 1999 (25) Choi et al., 2003 (26) Yamanishi et al., 1988 (27) Song et al., 1992 (28) Zhu et al., 1994 (29) Yongxin et al., 1998 (30) Yongxin et al., 1998 (30) Yongxin et al., 1998 (30)

References

908 Hammerbeck and Hooper

Virus-like particle/ chimeric viruses Inactivated virus

SEOV

Mouse Bank vole

DNA vaccine Np E. col–derived ANDV Np þ adjuvant

Hamster Rabbit Hamster Rhesus Cynomolgus Rhesus

Some No (Gn) /Yes (Gc) Not tested Some (PUUV) No Yesb No Yesa Yesa Yesa

Deer mouse Deer mouse

DNA vaccine Np DNA vaccine Gn or Gc peptides

Gn/Gc Gn/Gc Gn/Gc (HTNV þ ANDV) M gene M gene M gene (HTNV þ ANDV)

Yes No Some Some Yes Not tested No Not tested Yes Yes Yesa Yes (PUUV) Not tested

Gerbil Hamster Hamster Hamster Hamster none Hamster Mouse Hamster Hamster Rhesus Bank vole Deer mouse

Recombinant vaccinia expressed SEOV Gn/Gc Packaged Sindbis replicon Np Packaged Sindbis replicon Gn/Gc DNA-launched Sindbis replicon Np DNA-launched Sindbis replicon Gn/Gc Canine adenovirus type-2–expressed SEOV Gn DNA vaccine Np DNA vaccine Np DNA vaccine Gn/Gc DNA vaccine Gn/Gc DNA vaccine M gene E. coli–derived TOPV Np þ adjuvant Recombinant-cytomegalovirus expressed SNV Gn

vaccine vaccine vaccine vaccine vaccine vaccine

Most

Gerbil

DNA DNA DNA DNA DNA DNA

Not tested Not tested Not tested

Mosta Some Mosta

Human Hamster Human

Not tested Not tested No ANDV, SNV, BCCV Not tested ANDV, SNV, BCCV

PUUV, SEOV, SNV PUUV

Not tested Not tested

Not tested Not tested Not tested SEOV, HTNV Not tested Not tested PUUV, SEOV, SNV Not tested HTNV, SEOV, DOBV DOBV PUUV Not tested

SEOV, HTNV

PUUV HTNV, PUUV, DOBV Not tested HTNV, PUUV, DOBV

Yes (PUUV) Not tested Partial Not tested

Bank vole Mouse Mouse Mouse

Custer et al., 2003 (61) Hooper et al., 2008 (62) Hooper et al., 2006 (39) Custer et al., 2003 (61) Hooper et al., 2006 (39) Hooper et al., 2006 (30)

Lindkvist et al., 2007 (52) De Carvalho et al., 2002 (43)

Bharadwaj et al., 2002 (60) Bharadwaj et al., 2002 (60)

Xu et al., 1992 (55) Kamrud et al., 1999 (56) Kamrud et al., 1999 (56) Kamrud et al., 1999 (56) Kamrud et al., 1999 (56) Yuan et al., 2008 (57) Hooper et al., 1999 (58) Lindkvist et al., 2007 (52) Hooper et al., 1999 (58) Hooper et al., 2001 (38) Hooper et al., 2001 (38) De Carvalho et al., 2002 (43) Rizanov et al., 2003 (59)

Xu et al., 1992 (55)

Yongxin et al., 1998 (30) Yongxin et al., 1998 (30) Yongxin et al., 1998 (30)

De Carvalho et al., 2002 (43) Geldmacher et al., 2004 (34,35) Klingstrom et al., 2004 (54) Geldmacher et al., 2005 (36)

Johansson et al., 2002 (50) Bucht et al., 2001 (51) Bucht et al., 2001 (51) Lindkvist et al., 2007 (52) Hooper et al., unpublished Hooper et al., unpublished Maes et al., 2006 (53)

References

b

Based on plaque reduction neutralization test (PRNT) assay. Protects hamsters when passively transferred. Abbreviations: Np, N-protein; Gn, Gn glycoprotein; Gc, Gc glycoprotein; GHKC, golden hamster kidney cell; MUKC, Meriones unguiculatus kidney cell; TOPV, Topografov virus.

a

Recombinant protein

ANDV

DNA vaccine

Recombinant protein Virus-like particle/ chimeric viruses DNA vaccine

TOPV SNV

DNA vaccine

Virus-like particle/ chimeric viruses

Recombinant protein

Not tested Not tested Not tested PUUV, SEOV, SNV Not tested Not tested Not tested

Cross-reactivity

Not tested Some Some Not tested Yes Yesa Yes

Protection

Mouse Mouse Bank vole Mouse Hamster Rhesus Mouse

DNA vaccines

Animal model

DNA vaccine–modified Np peptide DNA vaccine Np DNA vaccine Np DNA vaccine Np DNA vaccine Gn/Gc DNA vaccine M gene E. coli–derived DOBV Np linked to K. Pneumoniae rP40 E. coli-derived DOBV Np þ adjuvant Yeast-derived DOBV Np þ/ adjuvant DOBV Np þ/ adjuvant HBV chimera–expressed DOBV Np peptides þ/ adjuvant Formalin-inactivated GHKC cells Formalin-inactivated GHKC cells b-Propiolactone inactivated MGKC cells (HTNV þ SEOV) Recombinant vaccinia expressed SEOV Np

Source of immunogen

Type of vaccine

DOBV

Virus targeted

Table 3 (Continued )

Chapter 83: Hantavirus Vaccines 909

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activity than a fourfold higher dose of Hantavax. We are unaware of any human clinical trials using this cell culture derivative. In China, there are at least three cell culture derived, inactivated virus vaccines based on HTNV and SEOV licensed for use (28,30). In general, all three vaccines appear clinically safe and elicit good humoral immune responses as measured by indirect immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Titers of neutralizing antibodies could be detected in up to 100% of the vaccine recipients (28,66,69) but the protective efficacy of these vaccines isn’t clear. Epidemiologic data suggest that there are fewer cases of HFRS in areas where these vaccines have been used (69). On the other hand, there are still 20,000 to 50,000 cases of HFRS in China per year (67), leading some Chinese researchers to suggest that there is still no effective prophylactic vaccine directed at HFRS (41).

Recombinant Protein–Based Vaccines The hantavirus glycoproteins and N-protein are major targets of both B cells and T cells during the immune response to HPS and HFRS viruses (reviewed in Ref. 68). As such, several studies have demonstrated that vaccination with recombinant N-protein is able to generate N-protein-specific antibodies and N-protein-specific T-cell response in vaccinated mice, hamsters, and bank voles (31,34,35,42–44,54). Importantly, in all these studies, vaccination with the purified N-protein by the subcutaneous or intramuscular route elicited partial or complete protection against infection. More recent attempts to test the oral delivery of PUUV N-proteins produced in transgenic plants have been less successful, likely due in part to degradation by proteases in the digestive tract (45). The addition of carrier proteins such as the outer membrane protein from Klebsiella pneumoniae (rP40), which facilitates uptake and crosspresentation of antigen (reviewed in Ref. 69), has been shown to improve the immunogenicity of Np vaccines (46,53), as has the addition of adjuvants such as heat shock proteins (32). Currently, however, there is only limited information regarding the efficacy of the Gn/Gc proteins as potential vaccine antigens. Hamsters actively immunized with recombinant Gn/Gc proteins were protected against challenge with HTNV, as were animals treated with passively transferred neutralizing monoclonal antibodies (31). In another report, deer mice vaccinated with plasmids encoding Gn peptides (compared to Gc or Np) showed the highest degree of protection from SNV infection and the best overall T-cell recall responses, but no neutralizing antibodies were detected (60). Aside from avoiding some of the inherent difficulties of working with hantaviruses to produce inactivated-virus vaccines, one advantage of recombinant N-protein vaccines is the high degree of N-protein homology (70) and the high degree of cross-reactivity of T-cell epitopes (71) and B-cell epitopes (52) in portions of the N-protein across hantavirus species. However, neutralizing antibodies target the hantavirus surface glycoproteins Gn and Gc, and there is no convincing evidence that antibodies to the N-protein or RdRp neutralize virus. Thus, it is likely that cell-mediated (i.e., T-cell-mediated) immunity plays a critical role in protection afforded by vaccination with purified N-protein. This, in fact, may be the biggest disadvantage of N-protein vaccines. There is evidence implicating the T-cell response to hantavirus infection in hantavirus disease pathogenesis, and how a more rapid and robust memory T-cell

response after vaccination may affect disease pathogenesis in the absence of a neutralizing antibody response is unknown.

Virus-Like Particles and Chimeric Virus Vaccines Virus-like particles (VLPs) have been explored as a vaccinology approach to multiple viruses (e.g., licensed human papillomavirus vaccine) due to their versatility and ability to generate robust immune responses in small animals, nonhuman primates, and humans. Researchers have had success using VLPs to protect against hantavirus infection. For example, chimeric hepatitis B virus (HBV) core particles containing various combinations of fragments of the PUUV, DOBV, and HTNV Nprotein protected bank voles against subsequent viral challenge (47,48) and induced cross-reactive N-specific antibody responses in mice (34,35). Other reports have described the successful use of hamster polyoma-derived VLPs (49) and vesicular stomatitis virus (VSV) pseudotypes (72) in mice to elicit anti-PUUV N-protein or HTNV Gn/Gc antibodies, respectively. While these approaches have been successful in preventing infection of rodents, none of these vaccines has been tested in nonhuman primates or lethal disease models.

Viral Live Vector Vaccines and Replicons A live recombinant vaccinia virus–based vaccine that expressed the HTNV S and M gene products (i.e., N-protein, Gn, and Gc) was able to induce HTNV and SEOV neutralizing antibodies and to protect hamsters from subsequent viral challenge (31,37). Similarly, SEOV Np, Gn, and Gc expressed by a recombinant vaccinia virus were shown to completely protect Mongolian gerbils against challenge with SEOV and HTNV viruses (55). A recombinant vaccinia virus expressing the HTNV S and M gene products is the only candidate subunit molecular hantavirus vaccine to be tested so far in humans (73). While administration of the live-virus vaccine was able to elicit HTNV neutralizing antibodies in recipients that had never been vaccinated against or exposed to vaccinia virus, development of the vaccine was halted, because it was poorly immunogenic in persons who had previously been vaccinated with vaccinia virus (i.e., the smallpox vaccine). A VSV pseudotype containing the HTNV glycoproteins (Gn/Gc) induced Gn and Gc neutralizing antibodies and protected mice from challenge with HTNV (72). Data are limited on the protective efficacy conferred by other recombinant carrier viruses expressing hantavirus antigens such as cytomegalovirus (59,74) or canine adenovirus type-2 virus (57). The utility of packaged replicons or viruses that infect but do not replicate has also been tested. Of the two investigated, Sindbis virus (56) and Semliki forest virus (75,76), only Sindbis virus was used in a vaccination study, and was found to elicit partial protection.

DNA Vaccines DNA vaccines offer the advantages of stimulating strong humoral and cellular immunity (at least in small animal models), while avoiding many of the risks associated with live and inactivated virus vaccines. To date, DNA vaccine candidates have been designed to deliver both the S gene (N-protein) and M gene (glycoproteins) of various hantaviruses. Several groups have elicited responses against SEOV, PUUV, and SNV by the delivery of naked S gene DNA segments (51,52,56,58,60). In mice, administration of S gene segments from these viruses

Chapter 83: Hantavirus Vaccines

stimulated rises in antibody titers, activated cellular immunity, and in some instances, provided partial protection. Vaccination of hamsters with S gene segments from SEOV, in the form of naked DNA or as DNA-launched replicons, was also able to elicit hantavirus-specific antibodies but vaccination conferred little to no protection from subsequent SEOV infection (56,58). Genetically linking the full-length HTNV S gene to the heat shock protein 70 (HSP70) gene does improve the antinucleocapsid immune response but no protection data have been reported (77). In general, vaccines that deliver the M gene have shown greater efficacy in conferring protection, probably due to the higher levels of neutralizing antibodies elicited (38,39,58,61,62). In this respect, the experiment by Hooper and colleagues using a plasmid containing the SEOV M gene (58) was the first to demonstrate that high titers of hantavirus-neutralizing antibodies could be elicited by a DNA vaccine delivered by gene gun. Indeed, numerous studies have demonstrated that the passive transfer of antibodies generated from hantavirus infection or vaccination with M segment-based DNA vaccines can protect from subsequent parenteral and respiratory exposure (31,39,61,62,78–83). Consistent with this, the degree of protection conferred upon animals receiving S gene DNA is far lower (approximately 20%) than that of animals receiving M gene DNA (80%–100%) (56,58,60). Chimeric M gene fusion vaccines have been created (40,41) but their ability to protect has not been tested in animal models. An overarching goal of hantavirus vaccine research is to achieve a vaccine capable of offering heterologous protection against multiple hantaviruses. In this respect, vaccination with vaccines expressing the M gene segments of both HFRS and HPS hantaviruses has been able to offer some heterologous protection in rodents (38,56) and nonhuman primates (38). In both animal models, the highest degree of cross-reactivity was found among HTNV, SEOV and DOBV, with less crossreactivity against PUUV. The recent development of a PUUV M gene-based DNA vaccine that is able to elicit neutralizing antibodies against PUUV in nonhuman primates and hamsters (Hooper et al., unpublished observations), however, suggests that a single HFRS-associated hantavirus vaccine could be achieved by a DNA vaccine that combines HTNV and PUUV M genes. DNA vaccines also represent one of the few attempts to create a vaccine for HPS-associated viruses. Nonhuman primates vaccinated with an ANDV M gene DNA vaccine produced neutralizing antibody titers as high as 1:20,480, and the antibodies were able to cross-neutralize two other HPSassociated hantaviruses, SNV and Black Creek Canal virus (BCCV) (61). The same ANDV M DNA vaccine was also found to be highly immunogenic in rabbits, but sera from these vaccinated animals failed to cross-neutralize SNV or BCCV (62). Moreover, passively transferred antibodies from nonhuman primates and rabbits vaccinated with ANDV M DNA vaccines effectively prevented disease in hamsters challenged with ANDV by the intramuscular or intranasal route even when administered several days after challenge (61,62). This is an important finding because hamsters represent the only pathogenic hantavirus disease model that closely mimics the capillary leak syndrome in humans (84). However, whereas the SEOV, HTNV, and PUUV M DNA vaccines were all highly immunogenic in hamsters, the ANDV M DNA vaccine was not immunogenic in hamsters (i.e., no neutralizing antibodies, no antibodies as measured by immunofluorescence, and no pro-

911

tection) (61). The lack of immunity of the ANDV M DNA vaccine in rodents appears to be a negative dominant phenomenon as hamsters vaccinated with a DNA vaccine containing both the HTNV and ANDV full-length M genes in the same plasmid (pWRG/HA-M) did not produce ANDV or HTNV neutralizing antibodies in any of the eight vaccinated hamsters (39). In contrast, macaques vaccinated with the same pWRG/ HA-M vaccine developed neutralizing antibodies against both HFRS and HPS viruses (39). The mechanism of this species difference is not understood and raises concerns as to how accurately animal models will predict immunogenicity in humans.

CONCLUSIONS Efforts to develop hantavirus vaccines have been ongoing for more than 50 years. Inactivated virus vaccines are in use in Asia where hantaviruses have afflicted millions; however, the numbers of cases in many endemic regions remain unacceptably high. Thus, efforts to create a safe and effective vaccine continue, and have resulted in a wide range of molecular approaches (Table 3). Many of these approaches have elicited immunogenicity and protection in rodent infection models but only a few approaches (i.e., M gene–based DNA vaccines) have been tested in nonhuman primates and in disease models (e.g., hamster challenge). To date, there has been only one molecular hantavirus vaccine candidate (vaccinia-vectored Hantaan M and S vaccine) that has been tested for immunogenicity in clinical trials in humans (73). The species difference observed with the ANDV M gene-based DNA vaccine in hamsters versus non-human primates (61) illustrates that the immunogenicity of these molecular vaccines can be species-dependent. Thus, there is a need to move more molecular hantavirus vaccines into phase 1 clinical trials to determine their safety and immunogenicity in humans. Any future hantavirus vaccine that can be consistently manufactured in a stable formulation and that has its safety and immunogenicity documented in phase 2 trials will face a daunting task to fulfill additional requirements for licensure. In many regions where hantaviruses have caused suffering, there are too few cases to perform a classical randomized, controlled phase 3 clinical trial to prove efficacy against natural exposure to the virus. Creative approaches to move modern Hantavirus vaccines towards licensure will be needed, such as use of the FDA ‘‘Animal Rule’’ to provide evidence of efficacy.

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30. Yongxin Y, Zhiyong A, Zhihui Y, et al. Inactivated cell-culture Hantavirus vaccine developed in China. In: JF Saluzzo JF, dodet B, (eds). Emergence and Control of Rodent-Borne Viral Diseases (Hantaviral and Arenal Diseases). Veyrier-du-lac, Annecy, France: Elsevier, 1998:157–161. 31. Schmaljohn CS, Chu YK, Schmaljohn AL, et al. Antigenic subunits of Hantaan virus expressed by baculovirus and vaccinia virus recombinants. J Virol 1990; 64(7):3162–3170. 32. Li J, Li KN, Gao J, et al. Heat shock protein 70 fused to or complexed with hantavirus nucleocapsid protein significantly enhances specific humoral and cellular immune responses in C57BL/6 mice. Vaccine 2008; 26(25):3175–3187. 33. Lee BH, Yoshimatsu K, Araki K, et al. A pseudotype vesicular stomatitis virus containing Hantaan virus envelope glycoproteins G1 and G2 as an alternative to hantavirus vaccine in mice. Vaccine 2006; 24:2928–2934. 34. Geldmacher A, Schmaler M, Kruger DH, et al. Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, longlasting, and highly cross-reactive immune response in mice. Viral Immunol 2004; 17(1):115–122. 35. Geldmacher A, Skrastina D, Petrovskis I, et al. An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice. Virology 2004; 323(1):108–119. 36. Geldmacher A, Skrastina D, Borisova G, et al. A hantavirus nucleocapsid protein segment exposed on hepatitis B virus core particles is highly immunogenic in mice when applied without adjuvants or in the presence of pre-existing anti-core antibodies. Vaccine 2005; 23(30):3973–3983. 37. Chu YK, Jennings GB, Schmaljohn CS. A vaccinia virus-vectored Hantaan virus vaccine protects hamsters from challenge with Hantaan and Seoul viruses but not Puumala virus. J Virol 1995; 69(10):6417–6423. 38. Hooper JW, Custer DM, Thompson E, et al. DNA vaccination with the Hantaan virus M gene protects Hamsters against three of four HFRS hantaviruses and elicits a high-titer neutralizing antibody response in Rhesus monkeys. J Virol 2001; 75 (18):8469–8477. 39. Hooper JW, Custer DM, Smith J, et al. Hantaan/Andes virus DNA vaccine elicits a broadly cross-reactive neutralizing antibody response in non-human primates. Virology 2006; 347(1):208–216. 40. Hao H, Xiu L, Zehua Z, et al. Genetic immunization with Hantavirus vaccine combining expression of G2 glycoprotein and fused interleukin-2. Genet vaccines Ther 2008; 6:15. 41. Zhang FL, Wu XA, Luo W, et al. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments. Biochem Biophys Res Commun 2007; 354(4):858–863. 42. Lundkvist A, Vapalahti O, Plyusnin A, et al. Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein. Virus Res 1996; 45(1):29–44. 43. de Carvalho Nicacio C, Gonzalez Della Valle M, Padula P, et al. Cross-protection against challenge with Puumala virus after immunization with nucleocapsid proteins from different hantaviruses. J Virol 2002; 76(13):6669–6677. 44. Dargeviciute A, Brus Sjolander K, Sasnauskas K, et al. Yeastexpressed Puumala hantavirus nucleocapsid protein induces protection in a bank vole model. Vaccine 2002; 20(29–30):3523–3531. 45. Khattak S, Darai G, Rosen-Wolff A. Puumala virus nucleocapsid protein expressed in transgenic plants is not immunogenic after oral administration. Virus Genes 2004; 29(1):109–116. 46. Maes P, Clement J, Cauwe B, et al. Truncated recombinant Puumala virus nucleocapsid proteins protect mice against challenge in vivo. Viral Immunol 2008; 21(1):49–60. 47. Ulrich R, Koletzki D, Lachmann S, et al. New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge. J Biotechnol 1999; 73(2–3):141–153.

Chapter 83: Hantavirus Vaccines 48. Ulrich R, Lundkvist A, Meisel H, et al. Chimaeric HBV core particles carrying a defined segment of Puumala hantavirus nucleocapsid protein evoke protective immunity in an animal model. Vaccine 1998; 16(2-3):272–280. 49. Gedvilaite A, Zvirbliene A, Staniulis J, et al. Segments of Puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice. Viral Immunol 2004; 17(1):51–68. 50. Johansson et al., 2002. 51. Bucht G, Sjolander KB, Eriksson S, et al. Modifying the cellular transport of DNA-based vaccines alters the immune response to hantavirus nucleocapsid protein. Vaccine 2001; 19(28–29):3820–3829. 52. Lindkvist M, Lahti K, Lilliehook B, et al. Cross-reactive immune responses in mice after genetic vaccination with cDNA encoding hantavirus nucleocapsid proteins. Vaccine 2007; 25(9):1690–1699. 53. Maes P, Keyaerts E, Bonnet V, et al. Truncated recombinant Dobrava hantavirus nucleocapsid proteins induce strong, longlasting immune responses in mice. Intervirology 2006; 49(5): 253–260. 54. Klingstrom J, Maljkovic I, Zuber B, et al. Vaccination of C57/BL6 mice with Dobrava hantavirus nucleocapsid protein in Freund’s adjuvant induced partial protection against challenge. Vaccine 2004; 22(29–30):4029–4034. 55. Xu X, Ruo SL, McCormick JB, et al. Immunity to Hantavirus challenge in Meriones unguiculatus induced by vaccinia-vectored viral proteins. Am J Trop Med Hyg 1992; 47(4):397–404. 56. Kamrud KI, Hooper JW, Elgh F, et al. Comparison of the protective efficacy of naked DNA, DNA-based Sindbis replicon, and packaged Sindbis replicon vectors expressing Hantavirus structural genes in hamsters. Virology 1999; 263(1):209–219. 57. Yuan Z, Zhang X, Zhang S, et al. Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus. Biologicals 2008; 36(3):162–167. 58. Hooper JW, Kamrud KI, Elgh F, et al. DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection. Virology 1999; 255(2):269–278. 59. Rizvanov AA, van Geelen AG, Morzunov S, et al. Generation of a recombinant cytomegalovirus for expression of a hantavirus glycoprotein. J Virol 2003; 77(22):12203–12210. 60. Bharadwaj M, Mirowsky K, Ye C, et al. Genetic vaccines protect against Sin Nombre hantavirus challenge in the deer mouse (Peromyscus maniculatus). J Gen Virol 2002; 83(pt 7):1745–1751. 61. Custer DM, Thompson E, Schmaljohn CS, et al. Active and passive vaccination against hantavirus pulmonary syndrome with Andes virus M genome segment-based DNA vaccine. J Virol 2003; 77(18): 9894–9905. 62. Hooper JW, Ferro AM, Wahl-Jensen V. Immune serum produced by DNA vaccination protects hamsters against lethal respiratory challenge with Andes virus. J Virol 2008; 82(3):1332–1338. 63. Hooper JW, Li D. Vaccines against hantaviruses. Curr Top Microbiol Immunol 2001; 256:171–191. 64. Cho HW, Howard CR, Lee HW. Review of an inactivated vaccine against hantaviruses. Intervirology 2002; 45(4–6):328–333. 65. Lee HW, Chu YK, Woo YD, et al. Vaccines against hemorrhagic fever with renal syndrome. In: Saluzzo JF, Dodet B, eds. Emergence and Control of Rodent-Borne Viral Diseases (Hantaviral and Arenal Diseases). Veyrier-du-lac, Annecy, France: Elsevier, 1998. 66. Park K, Kim CS, Moon KT. Protective effectiveness of hantavirus vaccine. Emerg Infect Dis 2004; 10(12):2218–2220. 67. Fang L, Yan L, Liang S, et al. Spatial analysis of hemorrhagic fever with renal syndrome in China. BMC Infect Dis 2006; 6:77.

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68. Schonrich G, Rang A, Lutteke N, et al. Hantavirus-induced immunity in rodent reservoirs and humans. Immunol Rev 2008; 225(1):163–189. 69. Jeannin P, Magistrelli G, Goetsch L, et al. Outer membrane protein A (OmpA): a new pathogen-associated molecular pattern that interacts with antigen presenting cells-impact on vaccine strategies. Vaccine 2002; 20(suppl 4):A23–A27. 70. Tischler ND, Rosemblatt M, Valenzuela PD. Characterization of cross-reactive and serotype-specific epitopes on the nucleocapsid proteins of hantaviruses. Virus Res 2008; 135(1):1–9. 71. Van Epps HL, Schmaljohn CS, Ennis FA. Human memory cytotoxic T-lymphocyte (CTL) responses to Hantaan virus infection: identification of virus-specific and cross-reactive CD8(þ) CTL epitopes on nucleocapsid protein. J Virol 1999; 73(7):5301–5308. 72. Lee BH, Yoshimatsu K, Araki K, et al. A pseudotype vesicular stomatitis virus containing Hantaan virus envelope glycoproteins G1 and G2 as an alternative to hantavirus vaccine in mice. Vaccine 2006; 24(15):2928–2934. 73. McClain DJ, Summers PL, Harrison SA, et al. Clinical evaluation of a vaccinia-vectored Hantaan virus vaccine. J Med Virol 2000; 60(1): 77–85. 74. Rizvanov AA, Khaiboullina SF, van Geelen AG, et al. Replication and immunoactivity of the recombinant Peromyscus maniculatus cytomegalovirus expressing hantavirus G1 glycoprotein in vivo and in vitro. Vaccine 2006; 24(3):327–334. 75. Billecocq A, Coudrier D, Boue F, et al. Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool. Clin Diagn Lab Immunol 2003; 10(4):658–663. 76. Kallio-Kokko H, Leveelahti R, Brummer-Korvenkontio M, et al. Human immune response to Puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells. J Med Virol 2001; 65(3):605–613. 77. Li J, Ye ZX, Li KN, et al. HSP70 gene fused with Hantavirus S segment DNA significantly enhances the DNA vaccine potency against hantaviral nucleocapsid protein in vivo. Vaccine 2007; 25(2):239–252. 78. Arikawa J, Yao JS, Yoshimatsu K, et al. Protective role of antigenic sites on the envelope protein of Hantaan virus defined by monoclonal antibodies. Arch Virol 1992; 126(1–4):271–281. 79. Medina RA, Mirowsky-Garcia K, Hutt J, et al. Ribavirin, human convalescent plasma and anti-beta3 integrin antibody inhibit infection by Sin Nombre virus in the deer mouse model. J Gen Virol 2007; 88(pt 2):493–505. 80. Xu Z, Wei L, Wang L, et al. The in vitro and in vivo protective activity of monoclonal antibodies directed against Hantaan virus: potential application for immunotherapy and passive immunization. Biochem Biophys Res Commun 2002; 298(4): 552–558. 81. Yoshimatsu K, Yoo YC, Yoshida R, et al. Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins. Arch Virol 1993; 130(3–4): 365–376. 82. Zhang XK, Takashima I, Hashimoto N. Characteristics of passive immunity against hantavirus infection in rats. Arch Virol 1989; 105(3–4):235–246. 83. Klingstrom J, Stoltz M, Hardestam J, et al. Passive immunization protects cynomolgus macaques against Puumala hantavirus challenge. Antiviral Ther 2008; 13(1):125–133. 84. Hooper JW, Larsen T, Custer DM, et al. A lethal disease model for hantavirus pulmonary syndrome. Virology 2001; 289(1):6–14.

84 SARS Vaccines Kanta Subbarao Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.

INTRODUCTION In February 2003, physicians at a hospital in Hanoi, Vietnam, sought advice from the local WHO office regarding a patient who had presented with an unusual influenza-like illness (1). Dr Carlo Urbani, an infectious disease specialist who responded to the request soon notified the WHO of an outbreak of severe respiratory disease. In the ensuing weeks, it became clear that similar outbreaks were occurring in several locations including Hong Kong, southern China, and Canada, and that patients in diverse locations had stayed at the same hotel in Hong Kong. The syndrome was called severe acute respiratory syndrome (SARS) and was characterized by fever, chills or rigors, headache, and nonspecific symptoms such as malaise and myalgias, followed by cough and dyspnea (2,3). Respiratory tract disease progressed to acute respiratory distress syndrome requiring intensive care and mechanical ventilation in more than 20% of patients. Prolonged hospitalizations associated with complications were reported, and advanced age was an independent correlate of adverse clinical outcome and increased mortality. The outbreak was notable for spread in health care settings, affecting large numbers of health care workers, and for a rapid dissemination to distant parts of the world by infected travelers. SARS first emerged in Guangdong province, China, around November 2002, where many of the affected individuals had contact with the live game trade (3). By the time the outbreak was over in July 2003, the WHO recorded more than 8000 cases including 774 deaths. In an admirable example of international cooperation in the face of a crisis, the scientific and public health community developed a provisional casedefinition to identify cases, rapidly identified the etiologic agent as a coronavirus, and determined the genetic sequence of the virus and established quarantine and travel advisories to limit the spread of the virus (4). The virus was named SARScoronavirus (SARS-CoV). As soon as the virus was isolated in cell culture, efforts to develop diagnostic tests, to identify compounds with antiviral activity, and to develop monoclonal antibodies (MAbs) and vaccines began in laboratories all around the world. The availability of a large number of vaccine platforms, including some (e.g., DNA vaccines) that were based on the published sequence but did not require access to infectious virus, led to a novel situation in which vaccine development efforts were undertaken before correlates of protection were established. Most of the candidate vaccines were based on one of the prototype strains of the outbreak, such as Urbani, Tor2, HKU 39849, or Bj01. The demonstration that intranasally

administered SARS-CoV replicated in the lungs of experimentally infected BALB/c mice, and that the mice developed a neutralizing antibody response that protected them from reinfection (5) was an important observation that made it possible to envision the development of an efficacious vaccine. The observation that neutralizing antibody is protective was confirmed in other animal models, though proof that neutralizing antibody in humans will protect from reinfection is still lacking because previously infected individuals have not been reexposed to another outbreak of SARS.

THE VIRUS, GENETIC DIVERSITY, AND THE RESERVOIR SARS-CoV belongs to the family Coronaviridae, which includes three phylogenetically distinct groups of viruses that infect a wide range of species. Group 1 coronaviruses include feline and canine coronaviruses and the human coronavirus 229E. Group 2 coronaviruses include mouse hepatitis virus, bovine coronavirus, and the human coronavirus OC-43. Group 3 includes infectious bronchitis virus that only infects avian species. SARS-CoV was initially thought to belong to a distinct fourth group of coronaviruses (6). However, based on further phylogenetic analysis and the presence of conserved cysteine residues, SARS-CoV is now classified as an early split-off from group 2 coronaviruses (7). The two prototype human coronaviruses, OC-43 and 229E, which were known prior to the appearance of SARS-CoV, cause common colds. The genome of SARS-CoV is a positive sense RNA that is 29,740 bp in length and the organization shows features characteristic of coronaviruses. Two overlapping open reading frames (ORF1a and ORF1b) encompass approximately twothirds of the viral genome and provide the proteins needed for replication and transcription. The remaining 30 part of the genome encodes four structural proteins that are arranged in the same order in all coronaviruses: S, spike protein; E, envelope protein; M, membrane protein; and N, nucleocapsid protein. The structural protein region of the genome also contains several genes that encode additional nonstructural proteins known as ‘‘accessory genes.’’ When SARS originally appeared in China in 2002, there was a history of contact with or exposure to animals from a live wild-game market (3,8). Samples from apparently healthy Himalayan palm civets and raccoon dogs yielded a virus that was closely related to SARS-CoV (99.8% nucleotide homology), and 13% to 40% of people who were occupationally exposed to

Chapter 84: SARS Vaccines

915

Table 1 Characteristics of SARS-CoV Isolates from Humans 2002–2003 outbreak

2003–2004??

Characteristic

Early phase

Middle phase

Late phase

???

Source Person-to-person transmission Morbidity and mortality Representative strains Genetic characteristics

Wild animal markets Low

Index case in a hospital Yes, local spread in China Severe disease, high mortality CUHK-W1, GZ50, HSZ-Bb, HSZ-Cb Purifying selection

Index case in hotel M Global spread

Wild animal market No

Severe disease, high mortality Urbani, Tor2, Frankfurt-1, HZS2-Fb, NS-1 Stabilization of nonsynonymous mutation rate Some isolates had 415 bp deletion in Orf8

Mild disease

Mild disease, low mortality GZ02, GZ60, HGZ8L 1a Some isolates had a 29-bp insertion or 82-bp deletion at Orf8 Strong positive selection

GD03, GZ0401, GZ0402 Sublineage distinct from human and animal viruses of 2002–2003 29-bp insertion in Orf8

Source: From Refs. 2, 15–16.

these animals were seropositive for SARS-CoV, and 73% of traders primarily trading in civet cats were seropositive for the virus (9). These findings suggested that civet cats were the reservoir or served as a carrier of the virus. Subsequent studies confirmed that civet cats in the live animal markets were infected with a SARS-CoV-like virus but the virus was not recovered from more than 1000 civet cats from 25 farms that were identified as the source of the market animals (10). Civet cats that were experimentally infected with the human and civet cat SARS-CoV isolates developed clinical signs of disease (11). These observations suggest that although civet cats may be an intermediate amplifying host, they are not the natural reservoir of the virus. Two independent groups of researchers have found genetic evidence of a coronavirus that is closely related to SARS-CoV in Chinese horseshoe bats (genus Rhinolophus) (12,13); bats may be the natural reservoir and source of SARS-CoV. The WHO declared the SARS outbreak over in July 2003, when the last chain of transmission was broken. When events were reconstructed using molecular epidemiology tools, it was noted that SARS cases had occurred in three phases: the early, mid, and late phases (14). The early phase consisted of sporadic cases, possibly of zoonotic origin, the middle phase resulted from extensive local transmission of the virus in a Guangdong hospital, and global transmission to more than 30 countries was seen in the late phase (Table 1) (14,15). The severity of the outbreak and the extent of secondary spread differed in these phases of the outbreak, and the viruses that were isolated during the different phases of the outbreak had unique genetic features (Table 1) (14,16). The following winter, four cases of mild respiratory illness occurred in Guangdong province, China. There was a history of contact with animals that were served in a restaurant in the exotic animal market but there was no secondary spread to contacts. The viruses identified in 2004 were genetically more similar to the civet cat isolates than to the human isolates from the previous year’s outbreak (Table 1). The only cases of SARS that have occurred after these cases in early 2004 resulted from laboratory accidents (18), one of which resulted in secondary cases and a fatality. The level of genetic relatedness of the spike protein gene of SARS-CoV with that of closely related viruses isolated from civet cats is *99.8% (8,10), and with those identified in horseshoe bats is 79% to 80% (12). A characteristic 29-bp insertion in

Orf8 was seen in the animal viruses, and in the late phase of the SARS outbreak, some isolates had a larger 415-bp deletion in this region (Table 1) (2,8,14). Liu and colleagues identified three major clusters of civet CoVs (19) in China, one group of viruses was resistant to neutralization by antibodies in convalescent sera from SARS patients and to antibodies experimentally induced with SARS-CoV vaccines (19). Contradictory findings have been reported regarding the ability of human and animal SARS-CoV isolates and vaccines to induce cross-reacting antibodies (19,20). If SARS were to reappear from an animal reservoir, it would be more likely to resemble the animal virus rather than the prototype SARS-CoV, and this has important implications for vaccine design.

THE IMMUNE RESPONSE IN RECOVERED INDIVIDUALS SARS primarily affects the respiratory tract but there is evidence that the virus is present in other sites, particularly the blood stream and the gastrointestinal (GI) tract. An understanding of the immune response to infection can provide a framework for the design of vaccines. Although a number of studies have been conducted and published on the immune response of patients who became infected with SARS-CoV, a majority of the patients were treated with antiviral and immunosuppressive drugs, and the studies were generally not organized systematically, making it difficult to compare results. The general principles that were established from following patients who developed SARS are as follows: IgG antibodies (21) and neutralizing antibodies are detected about a week after the onset of symptoms, levels peak at about 20 to 30 days (22) and are sustained for up to 16 months (23). Severe disease was associated with a more robust IgG response (21,24). A memory T lymphocyte response specific for the SARS-CoV N protein can persist for up to two years (25).

PROTECTIVE IMMUNITY AND THE GOALS OF IMMUNIZATION The Contribution of the Structural Proteins of SARS-CoV to Protective Immunity When mice were vaccinated with cDNA constructs encoding S, N, M, or E proteins, T cell and ELISA antibody responses were demonstrated (26). However, the ability of these vaccines

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Subbarao

Table 2 Preclinical Evaluation of Candidate SARS Vaccines Safety Immunogenicity ELISA, neutralizing antibody, cellular immune responses; Evaluation in young and older animals Efficacy Protection from experimentally administered challenge with homologous virus and zoonotic strains Experimental challenge early and late after vaccination Assessment in two or more models Evaluation in young and older animals Parameters Quantitative virology in the lungs in all animal models Weight loss in older mice, activity in hamsters Pulmonary pathology in relevant models, e.g., older mice, hamsters, nonhuman primates Passive transfer of serum from vaccinated to naı¨ve animals to determine whether protection is transferred by antibodies

to elicit neutralizing antibody and to confer protective efficacy was not evaluated. Buchholz and colleagues (27) cloned and expressed the structural proteins S, M, N, and E alone and in combinations of S þ M þ E together, and M þ E in a chimeric bovine-human parainfluenza virus type 3 (BHPIV3) and evaluated their immunogenicity and efficacy in golden Syrian hamsters (27). Only hamsters that were immunized with the S or S þ M þ E expressing viruses developed SARS-specific neutralizing antibodies and were fully protected from replication of the SARS-CoV challenge virus in the lower respiratory tract. These data indicate that the S protein is the only significant protective antigen among the structural proteins (27).

Epitopes Identified in the S and N Proteins of SARS-CoV The genome of SARS-CoV encodes a 1255 amino acid type I membrane spike (S) glycoprotein, oligomers of which form large peplomers in the viral envelope. The S protein mediates binding to the host cell through its receptor, angiotensinconverting enzyme-2 (ACE-2). There are two subunits of the S protein; the S1 domain extends from residues 5 to 680 and the S2 domain from 681 to1255. The S1 subunit binds to ACE-2 while the S2 subunit plays an important role in fusion of the viral envelope with the target cell membrane (28). The receptorbinding domain (RBD) maps to amino acids 318–510 (29–31) within the S1 subunit. The S protein and its fragments elicit antibodies that can neutralize infectivity of SARS-CoV (32–35). When sera from vaccinated animals were compared in ELISA and neutralization assays, a good correlation was observed between the two, suggesting that the ELISA assay may be an acceptable surrogate for neutralizing activity (36). However, in an evaluation of human MAbs against SARS-CoV generated from peripheral blood mononuclear cells (PBMC) from individuals who recovered from SARS, Traggiai et al identified several nonneutralizing antibodies that bound well in ELISA (37). T-cell epitopes have been identified on the S and N proteins of SARS-CoV. Using overlapping peptide pools tested as a peptide boost following DNA priming, S435–444 was identified as an H-2d restricted CD4 epitope and S365–374 as an H-2d restricted CD8 epitope in the S protein (38). Using a DNA vaccine expressing the N protein in B6 mice, an H2Db restricted CTL epitope was identified across amino acids 346–354 (39) and immunodominant N-specific ELIspot responses were elicited by five overlapping peptides N76–114 (40).

The Goal of SARS Vaccines An ideal SARS vaccine would prevent infection and/or disease caused by SARS-CoV. However, as discussed above, the virus associated with the greatest morbidity and mortality in 2002 to 2003 has not reappeared (Table 1), and if SARS-CoV or a closely related virus were to reappear, the likely source would be an animal host. Therefore, an ideal SARS vaccine in the period after 2003 should be effective against SARS-CoV isolates from the outbreak in 2002 to 2003, as well as those that resemble SARS-CoV-like viruses isolated from civet cats and horseshoe bats. However, the fact that few zoonotic strains of SARS-CoV have been isolated and maintained in cell culture presents a challenge in assessing the efficacy of candidate SARS vaccines against zoonotic strains (15). Synthetic biology and reverse genetics techniques have been used to generate recombinant isogenic viruses bearing variant spike glycoproteins derived from animal sources (15). These viruses will be very valuable for testing vaccine efficacy against heterologous challenge viruses in animal models (15). The safety and immunogenicity of candidate vaccines can be evaluated in experimental animals, and promising vaccines can be tested in clinical trials. Because SARS has not recurred, the efficacy of candidate vaccines will have to be inferred from studies in experimental animals. Table 2 outlines an approach to the preclinical evaluation of candidate SARS vaccines.

ANIMAL MODELS FOR EVALUATION OF SARS-CoV VACCINES Several laboratory animals support replication of SARS-CoV, but no single model replicates the disease seen in humans (41). The incubation period and duration of replication of SARS-CoV in animal models is shorter than in human cases of SARS, and the virus is generally cleared without causing fatal disease, even when infection is associated with pneumonitis, as it is in hamsters and older mice. Young mice do not develop clinical signs of illness and the virus is cleared by day 5 (5). In contrast, the virus replicates in the lungs of 12- to 14-month-old mice to higher titer and for a longer duration and is associated with transient weight loss and pneumonitis (42). SARS-CoV Urbani was adapted by several passages in BALB/c mice to become lethal for young mice (43). Transgenic mice expressing the human ACE-2 receptor develop neurologic disease following infection with SARSCoV, which was not a notable element of SARS in humans, so

Chapter 84: SARS Vaccines

there may be no added advantage to use of these animals for evaluation of SARS vaccine efficacy (44,45). Golden Syrian hamsters support efficient and prolonged (10–14 days) replication of SARS-CoV in the respiratory tract associated with decreased activity, pneumonitis, and pulmonary consolidation (46) permitting evaluation of several objective criteria. Ferrets also support replication of SARS-CoV in the respiratory tract following intranasal (IN) inoculation (47). Pulmonary virus replication is associated with histopathologic findings (47) but the extent of associated clinical symptoms is controversial (48,49). Cynomolgus and rhesus macaques, African green monkeys, and common marmosets have all been experimentally infected with SARS-CoV (50–55). The virus can be recovered from the lungs and is associated with histopathologic findings (52,53), but the extent of associated clinical symptoms is very variable and the virus is cleared quickly (53). Although some features of the disease in nonhuman primates are similar to what was seen in SARS cases in humans (50,54,55), nonhuman primates do not replicate SARS in humans faithfully (51,53), and the cost of studies in monkeys severely limits the size of experimental groups (50–55). Therefore, studies in nonhuman primates should be based on larger experiments in small animals and should be designed to answer specific questions.

ANTIBODY DEPENDENT ENHANCEMENT IN VITRO AND IN VIVO One of the major safety concerns about a SARS-CoV vaccine is regarding the development of antibody dependent enhancement (ADE) of disease. ADE has been associated with many different viruses. It occurs when a virus-antibody complex interacts with Fc receptors (FcRs) or complement to trigger virus uptake or alternatively, when antibodies induce conformational changes in envelope glycoproteins that are required for virus-cell membrane fusion (56). Initial concerns about ADE following SARS-CoV vaccines were based on the observation that accelerated and enhanced disease occurred on reexposure to feline infectious peritonitis virus (FIPV) in seropositive cats. Infection of macrophages by FIPV is believed to be important in the pathogenesis of accelerated disease; the enhanced disease was mediated by enhanced entry of FIPV into macrophages through anti-S antibodies binding to FcR expressed on the macrophages (57,58). Additional concerns about ADE associated with SARSCoV are related to the following four reports: (i) Entry of pseudotyped lentiviruses expressing the S protein of a SARSCoV isolated from a palm civet into a human renal epithelial cell line was enhanced by human S–specific neutralizing antibodies (59). (ii) Although SARS-CoV primarily infects epithelial cells in the lungs of mice, hamsters, and nonhuman primates, there is evidence of infection in some macrophages in nonhuman primates as well (53). (iii) Sera from mice and hamsters immunized with a recombinant native full-length trimeric spike protein vaccine and convalescent human sera showed a 100- to 1000-fold increase in virus entry into FcgRII positive, ACE-2-negative human B cells; this was mediated by the Fc region of the antibody and the FcgRII receptor (56). (iv) When ferrets that were immunized intraperitoneally with a modified vaccinia virus expressing the S or N protein of SARS-CoV were challenged with SARS-CoV, they were not protected. The ferrets were sacrificed on 27 to 29 days later, and all the animals had periportal and panlobular hepatitis with the most severe

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hepatitis with focal liver cell necrosis seen in animals that had been vaccinated with the modified vaccinia virus Ankara (MVA)-S vaccine (48,60). Although these reports raise some questions, the first report involves an assay of entry of a pseudotyped virus but has not been extended to use of an authentic SARS-CoV or the presence of an in vivo correlate of the enhanced entry of the pseudotyped virus. The report of enhanced entry into human B cell lines was not seen in mouse macrophages, despite the presence of FcgRII and B cells were only occasionally infected in SARS patients. Also, the trimeric spike vaccine elicited a protective immune response in vivo in hamsters (56). Neither of these studies is consistent with the ADE seen following FIPV vaccine. In the case of the MVA-S vaccine-induced hepatitis in ferrets, all the ferrets developed hepatitis and viral antigen was not detected in the liver. Also, MVA-S vaccines evaluated by two other groups of investigators were efficacious. Additionally, in a separate study in which ferrets were immunized with a weak inactivated vaccine, there was no evidence of enhanced disease (49). There are several studies in experimental animals in which sub-neutralizing levels of antibodies were present when the animals were experimentally infected with SARSCoV, but the animals did not show signs of enhanced viral replication or disease (5,49,61,62). On the basis of these observations, it is reasonable to conclude that SARS-CoV vaccines are not associated with ADE, as described with FIPV. However, as discussed below, there is reason to be cautious about vaccines that contain the N protein of SARS-CoV. An attenuated Venezuelan equine encephalitis virus expressing the N protein of SARS-CoV (VRP-N) failed to protect mice from homologous and heterologous SARS-CoV challenge and resulted in enhanced immunopathology with eosinophilic infiltrates in the lungs of mice after challenge. This pathology presented at day 4, peaked at day 7, and persisted through day 14, and was likely mediated by cellular immune responses in the absence of effective neutralizing antibody response (63).

PASSIVE IMMUNIZATION From previous studies of other coronaviruses and from early investigations of SARS in animal models, it became clear that neutralizing antibodies generated following infection are directed at the spike protein, and neutralizing antibodies could prevent replication of the SARS coronavirus in the lungs of mice. Convalescent plasma was administered to patients with SARS without adverse effects (64,65) but the benefit of this treatment strategy cannot be assessed because it was not a controlled clinical trial. MAbs represent an ideal alternative to hyperimmune sera. Strategies for generation of MAbs for use in humans include: (i) humanization of murine MAbs through protein engineering, (ii) selection of antibodies from phagedisplay libraries of human antibody fragments, and (iii) immunization of transgenic mice carrying human immunoglobulin loci followed by production of MAbs using the hybridoma technology. The MAbs that had potent in vitro neutralizing activity were able to prevent infection in mice (37,66,67), hamsters (68), and ferrets (69), and at least two human MAbs have been identified that cross-react with human SARS-CoV and zoonotic isolates in vitro and in vivo (70). Postexposure treatment with a MAb alleviated virus burden and the degree of associated pathology, including interstitial pneumonitis and consolidation in the hamster model (68).

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Use of MAbs for postexposure prophylaxis may be worth considering for specific high-risk groups such as health care workers (71) or household contacts of sporadic cases of SARS that are exposed before a diagnosis of SARS is entertained or in the event of laboratory exposure. MAb combinations (70,72) may also be an important adjunct to supportive treatment for SARS because specific antiviral therapy for SARS-CoV is not available.

ACTIVE IMMUNIZATION Several vaccine platforms that were explored to induce protective immunity against SARS-CoV including inactivated virus vaccines, subunit vaccines, vectored vaccines, live attenuated vaccines, DNA vaccines, and virus-like particles (VLPs) are outlined in Table 3 and are discussed in detail in the following sections. The goal of immunization is to induce humoral and/or cellular immunity to SARS-CoV. The safety, immunogenicity, and protective efficacy of SARS-CoV vaccines were evaluated in animal models (73). On the basis of promising findings in preclinical studies, a few vaccines have been evaluated in clinical trials.

VACCINE STRATEGIES Inactivated SARS-CoV Vaccines When it is possible to grow a virus to high titer in an acceptable substrate, a vaccine can be generated that consists of whole virus particles of which the infectivity is destroyed by formalin, or b-propiolactone (BPL) treatment, or exposure to ultraviolet light. This was the case with SARS-CoV, because it could be amplified in Vero cells. SARS-CoV was inactivated using BPL (61,74–77), formalin (49,78–81), ultraviolet treatment (82), or a combination of two inactivation strategies (83), and the vaccines were evaluated in mice (74–80,82,83), rabbits (75), ferrets (49), and nonhuman primates (61,81). The vaccines were administered to experimental animals by a variety of routes including subcutaneous (76,77,80,82), subcutaneous followed by a boost via the celiac route (78), intradermal (ID) (75), IN (80), intramuscular (IM) (61,81), or intraperitoneal (IP) (74), with a variety of adjuvants including complete and incomplete Freund’s adjuvant (75,78), aluminum hydroxide (61,74,77–80,82), MF-59, an oil squalenein-water emulsion (76), CpG (78), or cholera toxin B (80). In general, the vaccines elicited an IgG response in experimental animals and an IgM and IgA response was reported in some studies. A dose response was observed in the antibody titer, with higher doses of vaccine eliciting higher titers of SARSspecific ELISA IgG and neutralizing antibodies in the serum, and additional doses of vaccine led to an increase in IgG but not in IgM antibody titers (78,79,81,82). With one exception (77), inclusion of an adjuvant resulted in higher IgG antibody titers, though the increase in titer was not always statistically significant (61,74,76,78,82). There was a clear correlation between the SARS-specific ELISA antibody response and the neutralizing antibody response (83). The cellular immune response and the IgG subclass response to some candidate vaccines was characterized; a UV-inactivated vaccine administered with alum and a BPL-inactivated vaccine adjuvanted with MF-59 resulted in a Th2 response in mice (76,82) while two doses of a double (BPL and UV) inactivated whole virus vaccine elicited a substantial Th1 and Th2 response in mice (83). A formalin inactivated whole virus vaccine elicited a Th1 response in rhesus monkeys (81).

Although the immunogenicity of several candidate vaccines was evaluated in experimental animals, data on protective efficacy was not available for many of them (74,75,78– 80,82). In some studies, inactivated SARS-CoV vaccines were demonstrated to be efficacious in protecting mice (76,77,83) and nonhuman primates (61,81) from experimental challenge infection with SARS-CoV. Spruth and colleagues summarized data from 168 mice immunized with an inactivated whole virus vaccine and found that a SARS-specific ELISA IgG titer of 1:25,600 or neutralizing antibody titer  1:114 resulted in 100% protection from challenge (83). See and colleagues compared a BPL inactivated whole killed vaccine with a recombinant adenovirus vaccine administered intramuscularly or intranasally to 129 Sv/Ev mice and found that the whole killed vaccine induced much higher levels of serum neutralizing antibodies and was more effective in reducing pulmonary virus replication than the adenovirus vaccines (77). Characterization of the immune response to the whole killed vaccine led the authors to conclude that humoral not cellular immune responses correlate with the ability of the whole killed vaccine to protect against pulmonary SARS-CoV replication (77).

Subunit Vaccines Subunit vaccines are composed of purified viral protein that is generally administered with an adjuvant. Most of the subunit vaccines for SARS consist of purified, expressed recombinant S protein because the S protein elicits a neutralizing antibody response, though there are also a few reports in which the nucleoprotein (N) was expressed and evaluated as a vaccine. As noted earlier, the receptor binding domain and immunodominant epitopes of the S protein were mapped to the S1 domain or N-terminal part of the S protein. Therefore, several investigators chose to generate a truncated form of the S protein. A major advantage of truncated forms of the S protein that lack the transmembrane domain is that the recombinant protein can be expressed in soluble form (36,84). The S protein has been expressed and purified from several sources, including baculoviruses (36,84,85), bacteria (86), and plants (87). Bisht et al., Zhou et al., and He et al. generated recombinant baculoviruses expressing full length (36,85) or truncated forms of the SARS-CoV S protein (36,84,85). When administered to mice with adjuvants QS-21, saponin (84) or MPL þ TDM, Ribi adjuvant monophosphoryl lipid A (MPL) and trehalose dicorynomycolate (TDM) (84,85), aluminum hydroxide (36,88), or a proprietary adjuvant Protollin, recombinant purified truncated S proteins induced antibodies that recognized full-length membrane bound S protein by immunofluorescence (84) and truncated protein by ELISA (36,84,85), and neutralized infectivity of SARS-CoV (36,84,88) or of pseudovirions expressing the spike protein of human and animal SARS-CoV strains (85). A 1000- to >10,000-fold reduction in virus titer in the upper respiratory tract, and a reduction of more than a million-fold in virus titer in the lower respiratory tract was seen with the subunit vaccine generated by Bisht and colleagues (84). Although the bat SARS-like CoV has not been isolated in culture, the sequence of the virus is known and baculoviruses expressing the N and S proteins of the bat SARS-like CoV have been generated. ELISA antibody and cellular immune responses to SARS-CoV were elicited when mice were immunized subcutaneously or intraperitoneally. Unfortunately, neutralizing antibody titers and the protective efficacy of the

Preclinical evaluation in

Mice, rabbits, ferrets, nonhuman primates

Young and old mice, hamsters

Young and old mice, hamsters, ferrets, nonhuman primates

Hamsters

Mice

Type of vaccine

Inactivated virus vaccine

Subunit vaccines

Vectored vaccines

Live attenuated vaccines

DNA vaccines

A virus in which expression of the E protein gene was abolished was attenuated in hamsters but induced neutralizing antibodies against homologous and heterologous challenge viruses Vaccines expressing the S protein induced neutralizing antibodies and T-cell responses Vaccines expressing the N protein induced ELISA antibodies and T cell responses but no neutralizing antibodies

Both full-length and truncated S protein elicited an ELISA and neutralizing antibody response but two doses of S protein with adjuvant were required to elicit a neutralizing antibody response. Nt Ab titer increased with additional doses of vaccine and with increasing antigen. The inclusion of adjuvant enhanced antibody titers several fold. Antibodies generated in mice crossneutralized human and animal SARS-CoV Vaccines expressing the S protein induced neutralizing antibodies and T-cell responses; MVA-S induced a short-lived antibody response Vaccines elicited lower antibody titers in senescent mice than in young mice Intranasally administered vaccines elicited mucosal antibodies

Vaccines elicited an IgG and neutralizing antibody response A dose response was observed in the antibody titer Inclusion of an adjuvant resulted in higher IgG antibody titers

Immunogenicity

Table 3 Vaccine Strategies That Have Been Evaluated for SARS

Elicits an immune response most similar to natural infection

Does not require infectious virus

Genetic stability and recombination but strategies have been devised to diminish these risks

The foreign protein can be expressed in vivo in large quantities and the protein is often presented to the immune system in a natural form. Some vectored vaccines can be administered intranasally to elicit a mucosal immune response

Vaccines expressing the S protein conferred protection from challenge; some vaccines conferred protection from heterologous challenge as well as homologous challenge Efficacy was seen when animals were challenged soon after or months after immunization Expression of the N protein alone did not confer protection The vaccine conferred protection from homologous and heterologous challenge

Vaccines expressing the S protein conferred protection from challenge that was mediated by antibodies.

MVA-S immunized ferrets and VRP-N immunized mice developed enhanced pulmonary immunopathology on challenge

Protective antigen can be administered but antigens that do not play a role in protective immunity or induce immunopathology can be excluded

Vaccines administered with adjuvants offered protection from challenge. Specific lung IgA titers may be important in protection

Infectious virus has to be grown in large quantities for manufacture and requires special biocontainment facilities. The process of inactivation may alter certain proteins leading to an aberrant immune response The protein may not be expressed in native form

Vaccines contain several viral proteins. Vaccines can be generated even before the protective antigens are identified. The destruction of viral infectivity renders vaccines safe.

Vaccines that elicited neutralizing antibodies were efficacious in protection from challenge Humoral, not cellular immune responses correlated with protective efficacy

Concerns

Advantages

Efficacy

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S protein of the bat SARS-like CoV against SARS-CoV challenge were not reported (89). Kam and colleagues generated a codon-optimized recombinant native full length S protein fused to a C-terminal FLAG tag that when immunopurified contained correctly folded trimeric S protein (56). The trimeric spike protein vaccine (triSpike) was highly immunogenic in mice and hamsters. The addition of alum resulted in a higher titer and longer lasting neutralizing antibody response. The vaccine also elicited antigen-specific IgG and IgA in the intestine. triSpike immunization resulted in significant protection from SARS-CoV challenge in hamsters, and regardless of dose, triSpike immunized hamsters had reduced occurrence and severity of pneumonitis and no evidence of pulmonary consolidation or SARS-CoVassociated hepatic cellular necrosis. However, sera from mice and hamsters immunized with triSpike and convalescent human sera showed a 100- to 1000-fold increase in virus entry into FcgRII positive, ACE-2-negative human B cells. This antibody-dependent enhancement of virus entry was mediated by the Fc region of the antibody and the FcgRII receptor, but the significance of this finding is not clear for the reasons discussed in the section on ADE (56). The demonstration of an in vivo correlate of the in vitro enhanced entry into human B cells would warrant concern over the safety of this vaccine in humans. To induce a mucosal as well as systemic immune response to the S protein, Hu and colleagues intranasally administered baculovirus expressed S protein formulated with a proprietary adjuvant Protollin, which is Shigella flexneri lipopolysaccharide noncovalently associated with proteosomes composed of major outer membrane proteins of Neisseria meningitidis to mice (88). The Protollin-formulated S protein administered IN elicited a serum IgG antibody response, and antigen-specific IgA in lung lavage fluid was only detected in mice that received the Protollin-formulated S protein vaccine. A higher dose of Protollin resulted in a higher titer of IgG and IgA antibodies. Neutralizing antibody titers induced by both the adjuvanted vaccines were higher than titers induced by S protein alone. The Protollin-formulated S protein vaccine induced a balanced Th1/Th2 response or a bias toward a Th1 response while the alum-adsorbed S protein vaccine induced a Th2 response. The Protollin-formulated S protein vaccine and alum-adsorbed S protein induced similar levels of IgG and neutralizing antibodies in 12-month-old BALB/c mice but the titers were about three- to fourfold lower than titers in young BALB/c mice (88). When 12-month-old BALB/c mice were vaccinated with 30 mg of the Protollin-formulated S protein, they were completely protected from pulmonary virus replication, and this vaccine was better at protecting old mice from SARS-CoV infection than alum-adsorbed S protein vaccine. Both vaccines elicited comparable systemic immune responses but only the Protollinformulated S protein vaccine elicited mucosal immune response (88). Thus, specific lung IgA titers appear to be important in protection from SARS-CoV infection.

Vectored Vaccines The ability of viruses to efficiently infect cells, express their proteins, and stimulate an immune response has been exploited in the field of vaccinology. Attenuated viruses that have lost their ability to cause disease have been engineered to express foreign proteins. Vectored vaccines for SARS have been

generated using viruses from several families. Most of these viral vectors have been engineered to express the S protein, and some have been engineered to express the N protein as well. The cells that are targeted for expression of the foreign protein and the route of delivery of the vaccine depend on the viral vector used. For each of the vectored vaccines for SARS-CoV, the expression of the SARS protein was confirmed in vitro by Western blot or immunofluorescence, and the ability of the vaccine to induce a SARS-specific immune response was evaluated in experimental animals. Data on the protective efficacy in experimental animals are only available for a subset of the vaccines. Adenoviruses The S1, M, and N genes of SARS-CoV were expressed in a replication defective human adenovirus (huAd-5) or a chimpanzee adenovirus (77,90–92). The immunogenicity of the recombinant adenoviruses was evaluated in B6 and 129 Sv/ Ev mice (77,91), ferrets (77,91,92), and rhesus monkeys (90,92). Mice immunized with recombinant adenovirus (Ad-5) expressing the N protein of SARS-CoV developed an IgG1 and IgG2a ELISA antibody response to purified inactivated SARS-CoV and an antigen-specific cellular immune response (91). A recombinant adenovirus vaccine expressing the SARS-CoV S and N proteins (Ad S/N) administered intramuscularly or intranasally elicited neutralizing antibodies in mice, and the Ad S/N vaccine administered intranasally induced lower titers than the Ad S/N vaccine administered intramuscularly (77). However, the Ad S/N vaccine administered intramuscularly induced a higher cellular immune response, and Ad S/N vaccine administered intranasally was the only vaccine that induced an IgA response. Surprisingly, despite inducing high serum neutralizing antibody titers and a robust cellular immune response, the Ad S/N vaccine administered intramuscularly had only a limited effect in reducing pulmonary replication of challenge virus and although the Ad S/N vaccine administered intranasally induced lower serum neutralizing antibody titers and lower cellular immune responses, this vaccine provided protection from pulmonary virus replication. The fact that Ad S/N vaccine administered intranasally elicited an IgA response suggests that mucosal immunity contributed to its efficacy (77). In a separate study, rhesus monkeys immunized with two doses of recombinant adenoviruses expressing the S1, M, and N proteins of SARS-CoV developed SARSspecific antibody and cellular immune responses (90), but the ability of these vaccines to protect experimental animals from challenge was not reported. A single dose of a huAd-5 or chimpanzee AdC7 vaccine expressing a codon optimized S protein elicited a neutralizing antibody response in ferrets that was associated with a 100-fold reduction in titer of challenge virus and viral genome equivalents in the lungs (92). However, in both cases, reduction in pulmonary pathology was only modest. The neutralizing antibody response was greater in ferrets that were immunized with the huAd-5 vaccine and boosted with the AdC7 vaccine, and these animals had greater reduction in virus titer and significant reduction in pulmonary pathology. Although the authors specifically looked for it, there was no evidence of virus replication in the liver or spleen and no histopathology was reported in the liver (92). This prime-boost strategy with 13 weeks between priming with huAd-5 vaccine and boost with AdC7 was tested in four rhesus macaques. Neutralizing

Chapter 84: SARS Vaccines

antibodies were detected after the priming immunization and the titers boosted by 20-fold before they gradually declined. IFNg ELISPOTS using S peptide pools and intracellular cytokine staining showed that the T cell response to vaccination was primarily a CD8 response to multiple epitopes. The T and B cell responses in the macaques persisted at least 29 to 38 weeks. The protective efficacy of the vaccines was not evaluated in macaques (92). Alphaviruses An alphavirus, Venezuelan equine encephalitis virus, was engineered to express the S (VRP-S) or N (VRP-N) protein from the Urbani strain of SARS-CoV, and the vaccines were administered to young and senescent mice alone or in combination (VRP-S þ N) (63). The immunogenicity and efficacy of the vaccine were evaluated in mice against recombinant homologous and heterologous challenge viruses. In young mice, VRPS and VRP-S þ N induced similar titers against the homologous virus. Titers against the heterologous virus were low or undetectable even at five weeks after immunization. ELISA IgG titers in old immunized mice were reduced by 10-fold or more, compared with titers in mice that were immunized when they were young, and tested 12 months later. However, the ELISA antibody response to influenza hemagglutinin (control) followed a similar pattern and may reflect a general reduction in antigen-specific antibody production in older mice. Neutralizing antibody titers against homologous and heterologous viruses were low or undetectable when the mice were immunized when old (63). VRP-S and VRP-S þ N provided complete protection from IN challenge with SARS-CoV one month and one year after immunization of young BALB/c mice but VRP-N vaccine alone was not protective. VRP-S and VRP-S þ N provided only partial protection from heterologous challenge when mice were immunized at 6 to 12 months of age and were challenged 8 months later (63). Histopathologic examination of the lungs of VRP-S immunized mice was not remarkable. However, mice vaccinated with VRP-N showed marked bronchiolitis and alveolitis and interstitial accumulation of mononuclear leukocytes and eosinophils (63). Upon SARS-CoV challenge, VRP-S þ N immunized mice showed similar but less severe lymphoplasmacytic infitration around pulmonary vessels and bronchiolar airways. This pulmonary inflammatory response was not age dependent. The immunopathology was not due to antibody-induced enhancement but was likely mediated by cellular immune responses (63). From this experience, the investigators conclude that vaccines that induce a robust neutralizing antibody response against the homologous strain of SARS-CoV will likely confer protection against zoonotic introductions, especially in young populations, but vaccines that elicit complete protection against heterologous SARS-CoV in the young may not be sufficient for the elderly. They also urge cautious evaluation of any SARS-CoV vaccine containing the N protein (63). Paramyxoviruses A chimeric bovine parainfluenza type 3 virus that expresses the envelope proteins of human parainfluenza type 3 virus (BHPIV3) was engineered to express each of the structural proteins S, M, N, and E of SARS-CoV alone, or in combinations of S, M, and E or M and E (27,93). When the recombinant BHPIV3 viruses were administered to hamsters intranasally,

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the recombinant viruses replicated efficiently in the upper and lower respiratory tract, respectively (27). However, only hamsters that received the chimeric BHPIV3 viruses expressing S or S, M, and E proteins of SARS-CoV developed SARS-specific neutralizing antibodies and were protected from challenge with homologous SARS-CoV. Hamsters immunized with chimeric BHPIV3 viruses expressing the M, N, E or M and E proteins did not develop a neutralizing antibody response to SARS-CoV and were not protected from subsequent challenge, indicating that the S protein is the only significant protective antigen among the structural proteins of SARS-CoV (27). The chimeric BHPIV3/S was also restricted in replication in the lower respiratory tract of African green monkeys (93), but it induced moderate hemagglutination inhibition antibody titers against HPIV3 and a detectable neutralizing antibody response to SARS-CoV. Mucosal delivery of a single dose of the chimeric paramyxovirus was protective in a nonhuman primate model (93). The possibility of a low-level infection with the challenge virus cannot be ruled out in this study for two reasons. First, replication of the challenge virus was assessed using nose and throat swabs and tracheal washes but lung tissue was not examined, and it was later established that virus titers in nasal and tracheal swabs and washes do not accurately reflect the titer of virus in lung tissues in the African green monkey model (53), so the absence of virus in samples from the nose and throat do not completely rule out the possibility of pulmonary virus replication. Additionally, a rise in titer of SARS-CoVspecific neutralizing antibody was noted four weeks following challenge in the BHPIV3/S immunized monkeys that could have been induced by a low-level infection with the challenge virus (93). Poxviruses The highly attenuated MVA is a poxvirus that has accumulated numerous deletions and other mutations during the >500 passages in chick embryo fibroblasts, resulting in a severe host-range restriction that occurs at a late stage in viral assembly (62). Three groups of investigators generated MVA vaccines expressing the S protein (48,62,94) and N protein (48) of SARS-CoV. Bisht et al. immunized mice with MVA-S and detected ELISA antibodies against baculovirus expressed S1 protein and SARS-CoV-specific neutralizing antibodies (62). Mice that were immunized with MVA-S vaccine were fully protected from replication of challenge virus in the upper and lower respiratory tract. Serum antibodies elicited by the vaccine were sufficient to mediate protection because passive transfer of serum collected from mice immunized with MVA-S transferred protection to naı¨ve mice. There was no evidence of enhanced viral replication when mice with low titers of neutralizing antibodies were challenged with SARS-CoV (62). Chen et al. immunized mice, rabbits, and monkeys with an MVA-S vaccine and demonstrated that the vaccine elicited antibodies that neutralized infectivity of a pseudovirus expressing the SARS-CoV spike protein, indicating that MVA-S induces neutralizing antibodies without species restriction (94). The protective efficacy of the vaccine was evaluated in Chinese origin rhesus monkeys that received two doses of vaccine, half by the IM and half by the IN route. The monkeys were challenged with homologous SARS-CoV and euthanized seven days later; challenge virus was detected by RT-PCR in all and by virus isolation in some of the animals in the control group but not in MVA-S vaccinated monkeys; no evidence of enhanced disease was seen in the immunized monkeys (94).

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The experience reported by Weingartl and Czub on the evaluation of recombinant MVA vaccines in ferrets was quite different from the studies of Bisht et al. and Chen et al. (48,60,62,94). MVA-S and MVA-N vaccines were delivered by the intraperitoneal and subcutaneous route, to 6- to 10-weekold male ferrets, in two doses given two weeks apart. The MVA-N vaccine did not elicit a SARS-specific antibody response but the MVA-S vaccine elicited ELISA and neutralizing antibodies after two doses. However, the titer of neutralizing antibodies in MVA-S immunized ferrets declined rapidly and was not detectable at the time of challenge two weeks later when the ferrets received SARS-CoV Tor2 intranasally. After challenge, SARS-CoV-specific neutralizing antibody titers rose and peaked between seven and nine days postchallenge in MVA-S immunized ferrets and between days 19 and 21 in the other groups (MVA-N and MVA alone). However, this neutralizing antibody response did not protect the ferrets from replication or spread of the challenge virus. Virus was detected by PCR and virus isolation in pharyngeal swabs and blood, but there was no evidence of pulmonary disease on histopathologic examination. Serum alanine aminotransferase levels were elevated from days 5 to 21 after challenge in MVA-S immunized ferrets, and when the ferrets were euthanized between days 27 to 29, all the ferrets had a periportal and panlobular hepatitis that was most severe and associated with focal liver cell necrosis in two of the three ferrets that had been immunized with MVA-S. However, SARS-CoV antigen was not present in the liver tissue. The investigators urged caution in the development of SARS vaccines because vaccines expressing the SARSCoV N or S protein could enhance pathology and damage the liver and suggested that ferrets could be useful models for these studies. Because several aspects of this study were unusual, additional studies should be undertaken with this and other MVA-S vaccines to better understand its implications. For example, (i) the antibody response to the MVA-S vaccine was very short-lived in ferrets; two weeks after two doses of vaccine, none of the MVA-S vaccinated ferrets in this study had detectable neutralizing antibodies, and this observation was different from what was seen in mice, rabbits, and monkeys. (ii) All the ferrets developed hepatitis, though it was most severe in ferrets that received the MVA-S vaccine. It is not known whether the hepatitis was caused by SARS-CoV because viral antigen was not detected by IHC and the serum alanine aminotransferase elevation had resolved by the time the necropsy was performed. (iii) Some of the ferrets in the control group developed antibodies to SARS-CoV prior to challenge. (iv) This study has raised concerns about immunopathology following challenge in vaccinated animals, although the pathology was in the liver and not in the lung where the virus normally replicates following IN infection. It is not clear whether the short-lived neutralizing antibody response and hepatitis that was seen in ferrets was attributable to the use of MVA vectored vaccines, to ferrets, or both. Hepatitis was not seen in ferrets vaccinated with a weak inactivated SARS vaccine (49). Rhabdoviruses Two recombinant rhabdovirus vaccines have been engineered to express SARS-CoV proteins: an attenuated rabies virus and an attenuated vesicular stomatitis virus (VSV) (95,96). The goal of generating the recombinant rabies virus vaccine was to have a vaccine to immunize free-living wildlife, and for this approach to be effective, the vaccine has to be immunogenic

following a single oral dose (95). A highly attenuated rabies virus was used to express the S and N proteins of SARS-CoV. Although the vaccine was intended for oral administration, the vaccine was administered to mice by IM injection because mice were not ideal models for oral immunization. The recombinant vaccines induced antibodies to rabies virus, and the vaccine expressing the SARS-CoV S protein elicited a high titer of neutralizing antibody to SARS-CoV. The ability of these vaccines to protect experimental animals from challenge was not reported (95). The immunogenicity and protective efficacy of a recombinant attenuated VSV expressing the S protein was demonstrated in young BALB/c mice (96). Mice immunized with VSV-S were fully protected from replication of challenge virus in the upper and lower respiratory tract when challenged one month after immunization, and near complete protection was observed when mice were challenged four months after immunization. Although cellular immunity may contribute to the protective efficacy of this vaccine, antibody alone was sufficient to mediate this protection (96). In a separate study, 12- to 14-month-old BALB/c mice were immunized intranasally with the same VSV-S vaccine, and the immunogenicity and efficacy of the vaccine were evaluated one month after immunization (97). In contrast to the observation in young mice of a high titer neutralizing antibody response, the older mice that received the VSV-S vaccine had a detectable but a low titer antibody response. These older vaccinated mice were partially protected from weight loss and pulmonary virus replication on challenge. The titer of challenge virus in the lungs of VSV-S vaccinated mice was 10,000-fold lower than the titer in the mice that received the vector (VSV) alone; this difference was statistically significant (97).

Live Attenuated Vaccines A recombinant SARS-CoV was generated in which expression of the E protein gene was abolished (DE) in a SARS-CoV cDNA clone assembled as a bacterial artificial chromosome. The expression of the E protein was abrogated by the introduction of point mutations within the transcription-regulating sequence and at the start codon of the E gene. Additionally, to avoid the possibility of genetic reversion of the recombinant virus, a 142nt segment covering the majority of the E gene was deleted (98). The DE virus was attenuated in three different cell lines and was restricted in replication in the upper and lower respiratory tract of hamsters and was associated with reduced pulmonary inflammation (98) indicating that it may be a good candidate for a live attenuated virus vaccine (98). There may be additional mechanisms by which SARSCoV can be attenuated, and different approaches may be combined to increase the level of attenuation or to make the virus safer by making it ‘‘recombination resistant or recombination proof’’ (99,100). One such approach was to engineer a different transcription regulatory circuit into the genome that introduced genetic traps that were lethal in RNA recombination progeny viruses because mixed regulatory circuits in chimeric or recombined viruses promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated open reading frames and reduced expression of viral structural proteins (99). Another example was demonstrated in murine hepatitis virus (MHV A59), where the nsp1 was identified as a major pathogenicity determinant that interferes with the type I interferon system, and a virus with a 99 amino acid deletion in

Chapter 84: SARS Vaccines

nsp1 was severely attenuated in vivo but even low doses of the mutant virus elicited a strong CTL response and protected mice from homologous and heterologous virus challenge. If nsp1 is also a pathogenicity determinant in SARS-CoV, deletion of this gene could attenuate the virus (100).

DNA Vaccines On the basis of experience with animal coronaviruses, investigators focused on generating DNA vaccines expressing the S and N proteins of SARS-CoV. Yang et al. (101) generated two sets of cDNAs expressing the S protein using modified codons to optimize expression and to minimize recombination with endogenous coronaviruses; in one case the cytoplasmic domain (SDCD) and in another the transmembrane and cytoplasmic regions (SDTM) were deleted. Both cDNAs induced substantial humoral and cellular immune responses, and immunized mice were fully protected from pulmonary replication of challenge virus. Antibody was responsible for the protection conferred by these vaccines (101). Two groups of investigators generated DNA vaccines in which the N protein was linked to host cell proteins, calreticulin (CRT/N), and lysosome-associated membrane protein 1 (LAMP-1), respectively, with the intent to improve the immunogenicity of the N protein (39,40). CRT/N vaccinated mice had high ELISA antibody titers to recombinant GST-N protein and a H2Db restricted 9-mer peptide (amino acids 346–354) activated significantly more N-specific CD8+ T cells in splenocytes from mice vaccinated with CRT/N DNA than other putative epitopes. The LAMP-1 fusion led to higher IFNg T cell responses to the chimeric vaccine than N alone or than GST-N protein administered with complete Freund’s adjuvant (40).

Prime-Boost with DNA and Protein Vaccines A primary immunization with a DNA vaccine encoding the S protein was followed by a protein vaccine boost in the form of a whole killed virus vaccine, with or without an adjuvant or a recombinant expressed protein (102–104). Priming with a DNA vaccine followed by a boost with an inactivated virus vaccine produced a qualitatively different response than boosting with a recombinant adenovirus. The inactivated virus vaccine boost results in an increase in CD4+ responses and strong antibody responses, while the recombinant adenovirus boost leads to increases in CD8+ but not in CD4+ responses (103).

SARS-CoV Pseudoparticles (VLPs) VLPs were made by cotransfecting SARS-CoV M and N protein genes into 293 cells, with expression vectors expressing the four structural proteins of SARS-CoV. The M and N proteins were necessary and sufficient for the formation of intracellular pseudoparticles. The addition of S led to budding of particles with morphology (a corona) similar to SARS-CoV. However, these VLPs were not released efficiently (105), and until this problem is resolved, perhaps by changing the ratio of the proteins or by the addition of E, the use of this VLP vaccine strategy will be limited.

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cases that have occurred since then were mild or resulted from laboratory accidents. Thus, the need and demand for a SARS vaccine has not been large enough to justify the commitment of resources to take a vaccine to licensure. Concerns about the safety of a SARS-CoV vaccine linger, though ADE per se does not appear to be associated with SARS vaccines. Several candidate vaccines have been evaluated for safety and immunogenicity in animal models, and a few have been evaluated in clinical trials. Unless SARS reappears, the only efficacy data that will be available for candidate vaccines will be from preclinical studies in animal models. Although several animals can be experimentally infected with SARS-CoV, and the models reflect aspects of SARS disease in humans such as pneumonitis to varying degrees, none of the animal models mimics all aspects of SARS in humans or demonstrates the 10% mortality seen in SARS. Therefore, it is not clear whether the two-animal rule would apply for licensure of a SARS vaccine.

SUMMARY Studies in animal models suggest that vaccines that induce neutralizing antibodies are likely to be protective and that the spike protein induces neutralizing antibodies. If SARS reappears, it will be the result of an introduction of a SARS-like virus from an animal reservoir. Therefore, it will be critical to test the ability of SARS-CoV vaccines to induce antibodies that cross-react and neutralize SARS-like viruses that are identified in diverse animal species. Recombinant isogenic viruses bearing variant spike glycoproteins derived from animal sources will be very valuable tools for these determinations. Diminished immunogenicity in older animals was reported for three candidate SARS vaccines (63,88,97); these observations warrant attention because advanced age was a risk factor for severe disease and poor prognosis in SARS. An evaluation of vaccine efficacy in young and older mice should be undertaken whenever possible. Candidates for active or passive immunization for SARS include individuals who are at risk for exposure to SARS, as a result of their occupation such as health care workers, laboratory personnel, workers in live animal markets, or as a result of close household contact with newly identified cases of SARS. Although initial development of vaccines for SARS was very rapid and several candidate vaccines and MAbs show great promise in preclinical studies, the need and demand for SARS vaccines and immunoprophylaxis was not sustained long enough for these products to be evaluated in clinical trials to support an application for licensure. However, we can anticipate that the principles established from the vaccine research discussed above will form the basis for a rapid response, should the need for a SARS vaccine arise in the future.

ACKNOWLEDGMENTS This work was supported by funds from the Intramural Research Program of NIAID, NIH.

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85 Cancer-Specific Vaccines Drew Pardoll Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

INTRODUCTION Therapeutic vaccination for cancer continues to be a major approach to the overall immunotherapy of cancer. Historically, interest in cancer immunology stemmed from the perceived potential activity of the immune system as a weapon against cancer cells. In fact, the term ‘‘magic bullet,’’ commonly used to describe many visions of cancer therapy, was coined by Paul Erlich in the late 1800s in reference to antibodies targeting both microbes and tumors. Central to the concept of successful cancer immunotherapy are the dual tenets that tumor cells express an antigenic profile distinct from their normal cellular counterparts and that the immune system is capable of recognizing these antigenic differences. Support for this notion originally came from animal models of carcinogen-induced cancer in which it was demonstrated that a significant number of experimentally induced tumors could be rejected upon transplantation into syngeneic immunocompetent animals (1–4). Extensive studies by Prehn on the phenomenon of tumor rejection suggested that the most potent tumor rejection antigens were unique to the individual tumor (5). Since the original reports of Jenner over two centuries ago, prophylactic vaccination against infectious diseases has been one of the most influential medical interventions. Cancer vaccination, an immunotherapy approach applied to patients with established cancer, has tremendous potential based on the ability of both T cells and antibodies to specifically recognize cancer antigens and kill cancer cells expressing these antigens. However, at the time of this writing, human cancer vaccines have failed to yield an FDA approval in the United States despite multiple phase 3 clinical trials over the past two decades, although one vaccine, described in this chapter, is on a promising pathway to FDA approval. Despite the clinical failures of cancer vaccines to date, continuing molecular definition of tumor-specific and tumor-selective antigens, new vaccine platforms that selectively target and activate dendritic cells (DCs), and preclinical results with combinations of vaccination together with other immune modulators have generated renewed optimism that cancer vaccination will ultimately take its place among the pantheon of cancer therapies. As cancer genetics and genomics have exploded over the past decade, it is now quite clear that altered genetic and epigenetic features of tumor cells indeed result in a distinct tumor antigen profile. Overexpression of ‘‘oncogenic’’ growth factor receptor tyrosine kinases such as human epidermal growth factor receptor 2 (HER2/Neu) and epidermal growth factor receptor (EGFR) via epigenetic mechanisms has provided clinically relevant targets for one arm of the immune system—

antibodies (6,7). Indeed, monoclonal antibodies are the largest growing single class of cancer therapeutics based on successful new FDA approvals. In striking contrast, cellular immunotherapy of cancer has been quite disappointing in establishing therapeutic success in clinical trials to date. However, the history of monoclonal antibody development demonstrates that initial failures do not necessarily reflect a fundamental flaw in the general approach. The first successful phase 3 clinical trials with monoclonal antibodies came 15 years after initial clinical testing. The first-generation monoclonal antibodies either were fully murine or contained large components that were murine. Priming of human anti-mouse antibody responses in the patients resulted in clearance or therapeutic antibody upon repeat administration. Once humanized and fully human monoclonal antibody technologies were developed, this problem was solved. Additional engineering of the Fc regions of monoclonal antibodies to enhance antibody-dependent cellular cytotoxicity (ADCC) has provided even greater anticancer potency as these antibodies can recruit NK cells and macrophages into targeted tumors. Emerging insights about the nature of the interaction between the cancer and the immune system have led us to understand why cell-based cancer immunotherapy approaches such as therapeutic vaccines have been less potent against established cancer than originally imagined, providing hope that the failed clinical cancer vaccine trials of the past will give way to future clinical success, just as occurred with monoclonal antibodies. In general, we have learned that tumors employ mechanisms of tolerance induction to turn off T cells specific for tumor-associated antigens. Oncogenic pathways in tumors result in the elaboration of factors that organize the tumor microenvironment in ways that are quite hostile to antitumorimmune responses. Not only is the cancer capable of inducing potent tolerance among tumor-specific T cells, we now know that there are distinct forms of inflammatory and immune responses that are procarcinogenic. Thus, two frontiers in cancer immunology are the elucidation of how the tumor organizes its immune microenvironment as well as the nature of immune responses that are anticarcinogenic verses procarcinogenic. As the receptors, ligands, and signaling pathways that mediate immune tolerance and immune-induced procarcinogenic events are elucidated, these factors and pathways can be selectively inhibited by both antibodies and drugs in a way to shift the balance to antitumor immune responses. This chapter will outline the major features of tumor–immune system interactions and set the stage for molecularly based approaches to manipulate immune responses for successful cancer therapy.

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Table 1 Different Catagories of Human Tumor Antigens Category of tumor antigen

Advantages as vaccine antigen

Disadvantages as vaccine antigen

Arising from common oncogene/tumor suppressor gene mutation Examples Kras G12A (colon, pancreatic) BrafV599E (melanoma) P53 G249T (hepatoma) Cancer/testes antigens Examples Mage 1–12 (many tumors) NY-ESO-1 (many tumors) RAGE (renal, SSCHN, leukemia, others) GAGE (HNSCC, others)

l

Highly tumor specific Antigen-specific T-cell repertoire likely to be present Necessary for tumor growth/maintenance and therefore cannot be eliminated by tumor as an escape mechanism Highly tumor specific (only normal tissue expression is in testes) Many T-cell epitopes Shared among many different tumor types Tolerance to antigens may be limited

l

Upregulated in cancers via epigenetic mechanisms Examples CEA (gastrointestinal cancers) WT-1 (Wilms tumor, leukemias, lymphomas) Mesothelin (pancreatic, ovarian, mesothelioma) HER2/Neu (breast, ovarian cancers)

l

Shared among many tumors May be necessary for tumor growth/maintenance and therefore cannot be eliminated by tumor as an escape mechanism Some cell membrane antigens in this category may be additionally targets for monoclonal antibodies

l

Tissue-specific antigens shared by tumor Examples Tyrosinase (melanoma) MART1/MelanA (melanoma) gp100 (melanoma) PSA (prostate) Prostatic acid phosphatase (prostate)

l

Shared by many tumors Collateral damage to normal tissue counterparts may be highly acceptable for tumors derived from dispensable tissues (i.e., melanocytes, prostate)

l

Because they are self-antigens, immune tolerance may blunt vaccine-induced immune responses

Viral antigens expressed in tumors (or precancer) Examples Human papillomavirus E6, E7 (cervical cancer) EBV EBNA-1, LMP1,2 (Hodgkin’s, nasopharyngeal cancers)

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Highly tumor specific Large number of potential epitopes Antigen-specific T-cell repertoire likely present Necessary for tumor growth/maintenance

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Limited to tumors caused by viruses

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Highly limited epitopes (encompassing mutation site) available for HLA presentation in a given patient Limited number of examples of common mutations identified (but may increase with tumor genome sequencing efforts) Unnecessary for tumor growth/maintenance and therefore easily lost as an escape mechanism Heterogeneous expression within tumors, many tumor cells negative Not tumor specific. Collateral damage to normal tissues may result from strong induced immune response Because they are self-antigens, immune tolerance may blunt vaccine-induced immune responses

Abbreviations: EBV, epstein barr virus; HER2/Neu, human epidermal growth factor receptor 2; PSA, prostate specific antigen.

Indeed, adoptive T-cell transfer trials using ex vivo expanded tumor-specific T cells have demonstrated clear proof of principle that activated tumor-specific T cells can induce tumor regressions, even in patients with bulky metastatic cancer (8,9). Because adoptive T-cell transfer is prohibitively expensive, labor intensive, and extremely difficult to standardize, it is an immunotherapy approach unlikely to be broadly exportable. Most cancer immunotherapy efforts, including those that involve vaccination, seek to activate and expand tumorspecific T cells in vivo via various manipulations involving standardized reagents. The major barriers to be overcome are induction of tolerance among tumor-specific T cells and a tumor microenvironment that has developed to resist infiltration and attack by activated tumor-specific T cells. Although these two barriers represent significant hurdles to successful cancer immunotherapy, the elucidation of specific molecular mechanisms for tolerance induction as well as immune inhibition within the tumor microenvironment have lead to the generation of specific combinatorial approaches to cancer therapy (10).

CANCER ANTIGENS: THE DIFFERENCE BETWEEN TUMOR AND SELF Tumors reflect the biologic and antigenic characteristics of their tissue of origin but also differ fundamentally from their normal cell counterparts in both antigenic composition and biologic

behavior. Both these elements of cancer provide potential tumor-selective or tumor-specific antigens as potential targets for cancer vaccination specifically and antitumor immune responses in general (Table 1). Genetic instability, a basic hallmark of cancer, is a primary generator of tumor-specific antigens. The most common genetic alteration in cancer is mutation, which arises from defects in DNA damage repair systems of the tumor cell (11–15). Recent estimates from genome-wide sequencing efforts suggest that every tumor contains a few hundred mutations in coding regions (16). Additionally, deletions, amplifications, and chromosomal rearrangements can result in new genetic sequences resulting from juxtaposition of coding sequences not normally contiguous in untransformed cells. The vast majority of these mutations occur in intracellular proteins, and thus, the ‘‘neoantigens’’ they encode would not be readily targeted by antibodies. However, the MHC presentation system for T-cell recognition makes peptides derived from all cellular proteins available on the cell surface as peptide-MHC complexes capable of being recognized by T cells. On the basis of analysis of sequence motifs, it is estimated that roughly one-third of the mutations identified from genome sequencing of 22 breast and colon cancers (16) were capable of binding to common HLA alleles on the basis of analysis of sequence motifs (JP Allison, personal communication).

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In accordance with the original findings of Prehn (5), the vast majority of tumor-specific antigens derived from mutation as a consequence of genetic instability are unique to individual tumors. The consequence of this fact is that antigen-specific immunotherapies targeted at most truly tumor-specific antigens would, by necessity, be patient specific. However, there are a growing number of examples of tumor-specific mutations that are shared. The three best-studied examples are the Kras codon 12 G?A (found in roughly 40% of colon cancers and >75% of pancreas cancers), the BrafV599E (found in roughly 70% of melanomas) and the P53 codon 249 G?T mutation (found in *50% of hepatocellular carcinomas) (17–20). As with nonshared mutations, these common tumor-specific mutations all occur in intracellular proteins and therefore require T-cell recognition of MHC-presented peptides for immune recognition. Indeed, both the Kras codon 12 G?A and the BrafV599E mutations result in ‘‘neopeptides’’ capable of being recognized by HLA class I– and class II–restricted T cells (21–24). The other major difference between tumor cells and their normal counterparts derives from epigenetics (25). Global alterations in DNA methylation as well as chromatin structure in tumor cells result in dramatic shifts in gene expression. All tumors overexpress hundreds of genes relative to their normal counterparts and, in many cases, turn on genes that are normally completely silent in their normal cellular counterparts. Overexpressed genes in tumor cells represent the most commonly targeted tumor antigens by both antibodies and cellular immunotherapies. This is because, in contrast to most antigens derived from mutation, overexpressed genes are shared among many tumors of a given tissue origin or sometimes multiple tumor types. For example, mesothelin, which is targeted by T cells from vaccinated pancreatic cancer patients (26), is highly expressed in virtually all pancreatic cancers, mesotheliomas, and most ovarian cancers (27,28). While mesothelin is expressed at low to moderate levels in the pleural mesothelium, it is not expressed at all in normal pancreatic or ovarian ductal epithelial cells. The most dramatic examples of tumor-selective expression of epigenetically altered genes are the so-called cancertestis antigens (29). These genes appear to be highly restricted in their expression in adults. Many are expressed selectively in the testis of males and are not expressed at all in females. Expression in the testis appears to be restricted to germ cells, and in fact, some of these genes appear to encode proteins associated with meiosis (30–32). Cancer-testis antigens, therefore, represent examples of widely shared tumor-selective antigens whose expression is highly restricted to tumors. Many cancer-testis antigens have been shown to be recognized by T cells from nonvaccinated and vaccinated cancer patients (29). From the standpoint of immunotherapeutic targeting, a major drawback of the cancer-testis antigens is that none appear to be necessary for the tumors’ growth or survival. Therefore, their expression appears to be purely the consequence of epigenetic instability rather than selection, and antigen-negative variants are easily selected out in the face of immunotherapeutic targeting. A final category of tumor antigen that has received much attention encompasses tissue-specific antigens shared by tumors of similar histologic origin. Interest in this class of antigen as a tumor-selective antigen arose when melanomareactive T cells derived from melanoma patients were found to recognize tyrosinase, a melanocyte-specific protein required for melanin synthesis (33,34). In fact, the most commonly generated melanoma-reactive T cells from melanoma patients recognize

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melanocyte antigens (35). While one cannot formally call tissuespecific antigens tumor specific, they are nonetheless potentially viable targets for therapeutic T-cell responses when the tissue is dispensable (i.e., prostate cancer or melanoma). From the standpoint of T-cell targeting, tumor antigens upregulated as a consequence of epigenetic alterations represent ‘‘self-antigens’’ and are therefore likely to induce some level of immune tolerance. However, it is now clear that the stringencies of immune tolerance against different self-antigens differ according to tissue distribution and normal expression level within normal cells. The mesothelin antigen described earlier is such an example. In a recent set of clinical pancreatic cancer vaccine studies, mesothelin-specific T-cell responses were induced by vaccination with genetically modified pancreatic tumor cell vaccines and induction of mesothelin-specific T cells correlated with ultimate disease outcome. Given that the immune system is capable of differential responsiveness determined by antigen levels, it is quite possible to imagine generating tumor-selective immune responses against antigens whose expression level in the tumor is significantly greater within normal cells in the tumor-bearing host. Additionally, upregulated antigens that provide physiologically relevant growth or survival advantages to the tumor are preferred targets for any form of therapy since they are not so readily selected out. Beyond the antigenic differences between tumor cells and normal cells, there are important immunologic consequences to the distinct biologic behavior of tumor cells relative to their normal counterparts. Whereas uncontrolled growth is certainly a common biologic feature of all tumors, the major pathophysiologic characteristics of malignant cancer responsible for morbidity and mortality are their ability to invade through natural tissue barriers and to ultimately metastasize. Both of these characteristics, never observed in nontransformed cells, are associated with dramatic disruption and remodeling of tissue architecture. Indeed, the tumor microenvironment is quite distinct from the microenvironment of normal tissue counterparts. One of the important consequences of tissue disruption, even when caused by noninfectious mechanisms, is the elaboration of proinflammatory signals. These signals, generally in the form of cytokines and chemokines, are potentially capable of naturally initiating innate and adaptive immune responses. Indeed, the level of infiltration of leukocyte into the microenvironment of tumors tends to be significantly greater than that of the leukocyte component of their normal tissue counterparts. Cancers are therefore constantly confronted with inflammatory responses as they invade tissues and metastasize. In some circumstances, these inflammatory and immune responses can potentially eliminate a so-called tumor immune surveillance. However, as will be discussed, oncogenic pathways in the tumor not only appear to organize the immunologic component of the microenvironment in a fashion that protects the tumor from antitumor-immune responses but they can also qualitatively shift immune responses to those that actually support and promote tumor growth. It is these elements of the cancer–immune system interaction that will be the central targets of future immunotherapeutic strategies.

EVIDENCE PRO AND CON FOR IMMUNE SURVEILLANCE OF CANCER The fundamental tenet of the immune surveillance hypothesis, first conceived nearly half a century ago (36,37), is that a fundamental role of the immune system is to survey the body

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Figure 1 The balance between immune surveillance, resistance, and tolerance. Transformation of normal cells to cancer cells involves the creation of true neoantigens due to mutation as well as upregulation of self-antigens due to epigenetic forces. Successful immune surveillance of tumors based on recognition of these tumor-specific antigens would lead to tumor elimination at early stages. Clinically relevant tumor survival and progression require that tumors develop resistance mechanisms that inhibit tumor-specific immune responses to kill tumor cells. Alternatively, if the tumor develops mechanisms to induce immune tolerance to its antigens, antitumor effecter responses do not develop. Evidence is accumulating that tumors actively develop immune resistance mechanisms as well as immune tolerance mechanisms to survive despite displaying antigens capable of recognition by the immune system.

for tumors as it does for infection with pathogens, recognizing and eliminating them on the basis of their expression of tumorassociated antigens. In animal models, carcinogen-induced tumors can be divided into those that grow progressively (termed progressor tumors) and those that are rejected after an initial period of growth (termed regressor tumors) (1,2). The phenomenon of regressor tumors was thought to represent an example of the ongoing process of immune surveillance of cancer. A corollary to the original immune surveillance hypothesis is that progressor tumors in animals (presumed to represent clinically progressing cancers in humans) fail to be eliminated because they develop active mechanisms of either immune escape or resistance (Fig. 1). A fundamental prediction of the immune surveillance hypothesis is that immunodeficient individuals would display a dramatic increase in tumor incidence. After an extensive analysis of spontaneous tumor formation in immunodeficient nude mice, which have atrophic thymi and therefore significantly reduced numbers of T cells and T cell–dependent immune responses, no increased incidence of tumors was observed (38–42). These studies were taken as a major blow to the immune surveillance hypothesis. However, a caveat to the interpretation of these results is that nude mice still produce diminished numbers of T cells via thymus-independent pathways and can therefore mediate some degree of T cell–dependent immunity. In addition, nude mice frequently display compensatory increases in innate immunity that, as discussed in the following section, may represent a potent form of antitumor immunity and could contribute to immune surveillance of cancer. Epidemiologic studies of patients with heritable immunodeficiencies revealed a significantly increased risk of certain cancers that are distinct from the epithelial cancers commonly observed in normal immunocompetent adults (43–45). Many of these cancers are also observed in transplant patients on chronic pharmacologic immune suppression as well as in HIV/AIDS patients whose immune system is depressed. The most common cancers in these individuals include lymphoplastic lymphomas as well as Kaposi’s sarcoma; however, certain epithelial cancers, such as stomach cancer, were also observed at increased frequency. A unifying theme for the majority of cancers observed in immunodeficient individuals is their microbial origin. The

majority of lymhoplastic lymphomas are Epstein Barr Virus (EBV)-associated lymphomas (46), and Kaposi’s sarcoma is a result of infection with the herpesvirus Kaposi’s sarcoma herpesvirus (KSHV) (47). Other virus-associated cancers such as cervical cancer (from human papillomavirus) (48,49) are also observed at increased frequency. It is now appreciated that stomach cancer is associated with ulcer disease related to infection with the bacterium Helicobacter pylori (50). From these studies, the notion emerged that immune surveillance indeed protects individuals against certain pathogen-associated cancers by either preventing infection or altering chronic infection by viruses and other microbes that can eventually induce cancer. These studies were taken to represent evidence that the common non-pathogen-associated cancers most commonly seen in adults in developed countries (i.e., prostate cancer, colon cancer, lung cancer, etc.) are not subject to immune surveillance (51). Two caveats to this interpretation must, however, be noted. First, detailed epidemiologic analyses of immunodeficient individuals were performed at a time when these patients rarely lived beyond their 20s and 30s, when cancer incidence normally increases most significantly. It is therefore possible that a subtler cumulative increased incidence of common non-pathogen-associated cancers would have been observed had these individuals lived further into adulthood. Indeed, more recent analyses definitively demonstrate an increase in incidence of some non-pathogen-associated cancers in immunodeficient individuals, particularly melanoma (52,53). In addition to epidemiologic data, dramatic anecdotal examples are difficult to ignore. There have been a number of reports of donor-derived melanoma in immunosuppressed kidney transplant recipients even though the kidney donor had been in complete tumor remission at the time of transplant (54–56). These results indicate that at least for some non-pathogenassociated tumors, the immune system can play a significant role in maintaining the micrometastatic disease in a dormant state. Whether this principle applies to non-pathogen-associated human tumors besides melanoma remains to be demonstrated. A number of recent studies reevaluating tumor immune surveillance in genetically manipulated mice have revealed clear-cut evidence that various components of the immune system can at least modify, if not eliminate, both carcinogeninduced and spontaneously arising cancers. A series of studies

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by Schreiber and colleagues reexamined cancer incidence in mice rendered immunodeficient via genetic knockout of either the RAG2 gene (deficient in both B and T cells), the g-interferon (IFN) receptor gene, STAT1 gene, or the type 1 IFN receptor gene (57–59). When these knockout mice were either treated with carcinogens or crossed onto a cancer-prone P53 knockout background, the incidence of cancers was modestly but significantly increased relative to nonimmunodeficient counterparts when observed over an extended period (>1 year). Transplantation studies demonstrated that direct g-IFN insensitivity by the developing tumors played a significant role in the defect in immune surveillance. Interestingly, in contrast to g-IFN receptor knockout mice, the mechanism for increased tumor incidence in tumors in type 1 IFN receptor knockout mice did not involve sensitivity by the tumor to type 1 IFN but rather reflected role of the type 1 IFN in induction of innate and adaptive immunity. Even animals not crossed onto a cancerprone genetic background or treated with carcinogens developed an increased incidence of invasive adenocarcinomas when observed over their entire life span. Furthermore, g-IFN, RAG2 double knockout mice developed a broader spectrum of tumors than RAG2 knockout mice. All of the tumors that arise in these genetically manipulated immunodeficient animals behave as regressor tumors when transplanted into immunocompetent animals. These findings indeed suggest that tumors that arise in immunodeficient animals would have been eliminated had they arisen in immunocompetent animals. The relatively subtle effects on tumorigenesis, requiring observation over the life span of the animal, suggest that the original concept of immune surveillance of tumors arising on a daily basis is, in fact, not correct. Instead, it is clear that the presence of a competent immune system ‘‘sculpts’’ the tumor through a process that has been termed immunoediting. One of the caveats in the interpretation of these studies comes from the work of Dranoff and colleagues, who studied mechanisms of increased tumorigenesis in GM-CSF, g-IFN double knockout mice (60). While they observed an increase in gastrointestinal and pulmonary tumors, they noted that such animals harbored infection with a particular bacterium not normally observed in immunocompetent animals. Maintenance of these double knockout mice on antibiotics essentially eliminated the increased rate of tumor formation. Thus, some of the increased tumor rates in genetically immunodeficient animals could be related to unappreciated chronic infections that develop in these animals, which are not housed under germ-free conditions. Nonetheless, while the classic concepts of immune surveillance of cancer remain unsupported by experimental evidence, studies of tumorigenesis in genetically manipulated immunodeficient mice indeed suggest that developing tumors must actively adapt themselves to their immune microenvironment to exist within the context of a competent immune system.

INNATE IMMUNITY, EPITHELIAL IMMUNITY, AND TUMOR IMMUNE SURVEILLANCE While much emphasis has been placed on the role of adaptive immunity, particularly conventional T cells, in immune surveillance of cancer, a confluence of more recent findings points to innate immunity and epithelial immunity in the immunologic sensing of carcinogenic events in the skin, gut, and possibly other sites. Much of the evidence focuses on the natural killer receptor G2D (NKG2D) receptor. NKG2D was originally

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defined as an activating NK receptor (61–63). Most NK receptors appear to be inhibitory when engaged—this inhibition is often associated with immunoreceptor tyrosine kinase–based inhibitory motif (ITIM) domains in the cytoplasmic tails. ITIM provide docking sites for phosphatases that oppose the activity of tyrosine kinases involved in lymphocyte activation. NK activation status is a balance between engagement of activating and inhibitory receptors. NKG2D, the best-studied activating receptor on NK cells, is somewhat unusual in that it does not contain an immunoreceptor tyrosine kinase–activating motif (ITAM) and is associated with an adapter molecule, DAP 10, which contains neither conventional ITIM nor ITAM (64). Instead, DAP 10 contains a KYXXM motif that appears to bind to phosphotidyl innositol (PI) 3 kinase upon phosphorylation of the tyrosine in this motif. NKG2D is expressed on all NK cells as well as on some ab and gd T cells. Beyond NK cells, NKG2D is expressed at high levels on a number of subsets of intraepithelial lymphocytes (IELs). IELs represent a distinct population of lymphocytes residing in epithelial tissues that display features of both adaptive and innate immune responses (65–69). They are thought to represent a major first line of defense against pathogens attempting to invade across epithelial linings exposed to the environment (i.e., skin, gut, respiratory tract). Fifty percent of the IELs of the gut express the gd T cell receptor (TCR) (normally expressed by <3% of circulating T cells), while the other 50% express the common ab-TCR. gdTCR-expressing IELs in different compartments express a very restricted repertoire and are thought to recognize certain types of microbial antigens or potential self-antigens associated with stress or inflammatory responses to microbial infection. Even the ab-TCR-expressing IELs have an extremely restricted TCR repertoire similar to invariant natural killer T cells (NKT cells). A significant subset of gut IELs express a particular VaVb and are thought to recognize a limited subset of microbial or selfnonpeptide antigens presented by nonclassical class I MHC molecules. Thus, NKG2D expression marks diverse subsets of lymphocytes that, though expressing different families of recognition receptors, act as components of innate immunity in that they recognize a stereotypical set of antigens associated with infection or stress. The first evidence that the NKG2D receptor might play a role in tumor-immune surveillance came from the finding that normal colonic epithelium as well as a significant proportion of tumors could express the two defined human ligands for NKG2D—MICA and MICB. MICA and MICB, which represent nonclassical MHC class I type molecules whose structure demonstrates no antigen-binding grove characteristic of most MHC molecules, are stress-induced proteins whose genes contain stress response elements in their promoters (70,71). Raulet and colleagues have demonstrated that upregulation of MICA/B is induced through the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3 related (ATR)/checkpoint kinase 1 (Chk1) pathway of DNA damage recognition (72). An analysis in human cancer suggested a correlation between expression of MICA/B and infiltration of certain subsets of gd T cells that express NKG2D. Initially, it was proposed that MICA and MICB were direct ligands for specific gd receptors themselves as well as NKG2D (73,74), but this idea is controversial. MICA and MICB do not have any murine orthologs, but murine NKG2D does bind to products of the retinoic acid inducible gene family, retinoic acid early inducible-1 (RAE-1) a–e, as well as the product of the H60 gene. UL16 binding protein 3 (ULBP3) is an additional NKG2D ligand to be described (75,76). These

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NKG2D ligands appear to be involved in immune recognition and possibly tumor surveillance of mice (77–79). Recognition and killing of murine skin keratinocytes or intestinal epithelial cells by gd IELs require expression of NKG2D ligands and are blocked by anti-NKG2D antibodies. Transfection of murine tumors with genes encoding NKG2D ligands renders them susceptible to NKG2D-dependent killing by NK cells. Emerging data on NKG2D function on IELs together with the potentially stress-induced nature of its ligands suggests that the IEL system of immune surveillance may indeed be relevant to carcinogenesis as well as infectious challenges (79). The major initiating event of carcinogenesis in the skin—UV light—is a potent source of DNA damage, which, as mentioned earlier, has been shown to induce NKG2D ligands via the ATM pathway. Thus, in addition to endogenous killers of genome-damaged cells, such as P53, IELs, and NK cells may represent an extrinsic sensor of DNA damage and genotoxic stress via recognition of cells that have upregulated NKG2D ligands. As with the case of classic immune surveillance mediated by classical T cells, the emergence of a clinically evident cancer implies that the tumor has developed a mechanism to circumvent or evade any innate immune surveillance systems. In the case of the NKG2D system, Spies and colleagues have provided suggestive evidence that tumors can shed MICA/B in a soluble form as a means of evading NKG2D-dependent recognition. They demonstrated that certain tumors are associated with high levels of shed MICA/B and that soluble MICA/B binds to and downmodulates NKG2D on NK cells, thereby acting as an antagonist to NKG2D activation via cell surface–bound MICA/B (80). While this mechanism remains to be proven as a true evasion system for NKG2D-dependent tumor recognition, it points out the diversity of mechanisms that tumors utilize to evade immune recognition. It also points out straightforward approaches to block these evasion systems. If indeed soluble MICA/B does represent a mechanism for tumorimmune evasion of innate immune recognition, antibodies that would bind to and clear soluble MICA/B but not block the interaction between cell membrane MICA/B and NKG2D on NK cells could potentially restore the capacity of NK cells to recognize MICA/B-expressing tumors.

IMMUNE TOLERANCE AND IMMUNE EVASION: THE HALLMARK OF A SUCCESSFUL TUMOR While controversy over the ultimate role of immune surveillance in natural modulation of cancer development and progression will undoubtedly continue into the future, one can summarize the current state of knowledge as supporting the notion that natural immune surveillance plays a much smaller role than what was originally envisioned by Thomas and Burnet. However, developing tumors need to adapt to their immunologic milieu in a manner that either turns off potentially harmful (to the tumor) immune responses or creates a local microenvironment inhibitory to the tumoricidal activity of immune cells that could inadvertently become activated in the context of inflammatory responses associated with tissue invasion by the tumor. These processes—tolerance induction and immune evasion—have become a central focus of cancer immunology efforts and will undoubtedly provide the critical information necessary for development of successful immunotherapies that break tolerance to tumor antigens and break down the resistance mechanisms operative within the tumor microenvironment.

Evidence from both murine tumor systems as well as human tumors strongly demonstrates the capacity of tumors to induce tolerance to their antigens. This capacity to induce immune tolerance may very well be the single most important strategy that tumors use to protect themselves from elimination by the host’s immune system. Tolerance to tumors appears to operate predominately at the level of T cells; B-cell tolerance to tumors is less certain because there is ample evidence for the induction of antibody responses in animals bearing tumors as well as human patients with tumors. However, with the exception of antibodies against members of the EGFR family, there is little evidence that the natural humoral response to tumors provides significant or relevant antitumor immunity. In contrast, numerous adoptive transfer studies have demonstrated the potent capacity of T cells to kill growing tumors, either directly through cytotoxic T lymphocyte (CTL) activity or indirectly through multiple CD4-dependent effector mechanisms. It is thus likely that induction of antigen-specific tolerance among T cells is of paramount importance for tumor survival. The first direct evidence for induction of T-cell tolerance by tumors was provided by Bogen and colleagues, who examined the response of TCR transgenic T cells specific for the idiotypic immunoglobulin expressed by a murine myeloma tumor (81,82). They first demonstrated induction of central tolerance to the myeloma protein followed by peripheral tolerance. Using influenza hemagglutinin (HA) as a model tumor antigen, Levitsky and colleagues demonstrated that adoptively transferred HA-specific TCR transgenic T cells were rapidly rendered anergic by HA-expressing lymphomas and HAexpressing renal carcinomas (83,84). Tolerance induction has been demonstrated in both the CD4 and CD8 compartments. In general, initial activation of tumor-specific T cells is commonly observed; however, the activated state of T cells is typically not sustained with failure of tumor elimination as a frequent consequence. Tolerance induction among tumor antigen– specific T cells is an active process involving direct antigen recognition, although in some murine systems, tolerance to tumors appears to be associated with failure of antigen recognition by T cells, that is, the immune system ‘‘ignores’’ the tumor (85,86). Beyond studies on transplantable tumors, more recent analyses of immune responses to tumor antigens in tumor transgenic mice developing spontaneous cancer have further emphasized the capacity of spontaneously arising tumors to induce tolerance among antigen-specific T lymphocytes. In a model of prostate tumorigenesis, Drake and colleagues evaluated CD4 responses to HA and double transgenic animals expressing HA and SV40 T antigen under control of the prostate-specific probasin promoter (87). Development and progression of prostate tumors did not result in enhanced activation of adoptively transferred HA-specific T cells. Tolerance to HA as a normal prostate antigen occurred largely through ignorance since there was no evidence for antigen recognition by HA-specific T cells. However, increased recognition was observed upon either androgen ablation (which causes massive apoptosis within the prostate) or development of prostate cancer. Nonetheless, enhanced antigen recognition was not accompanied by activation of effector functions such as g-IFN production. Analysis of the consequences of transformation in additional tumor transgenic mouse systems has also been performed. Blankenstein and colleagues evaluated T-cell responses and rejection in a model of sporadic induction of tumors associated with expression of a tumor-specific antigen

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only at the time of transformation (88). They found that preimmunization of mice against the tumor-associated antigen prevented the development of tumors. However, nonimmunized mice developed spontaneous tumors without any significant evidence of natural immune surveillance in the absence of preimmunization. They further demonstrated that an initial antigen-dependent activation of tumor-specific T cells could be observed at the time of spontaneous tumor induction but that this recognition ultimately resulted in an anergic form of Tcell tolerance similar to that observed by Drake and colleagues in the prostate system. The capacity of spontaneously arising tumors to tolerize T cells has not been uniformly observed. A contrasting result by Ohashi and colleagues was observed when LCMV GP33–specific TCR transgenic CD8 T cells were adoptively transferred into double transgenic mice expressing both SV40 T antigen and LCMV GP33 under control of the rat insulin promoter (89). These animals develop pancreatic islet cell tumors that express GP33. These investigators found that as tumors progressed in the mice, enhanced T-cell activation occurred. CD8 T-cell activation was demonstrated through bone marrow chimera experiments to occur exclusively via cross-presentation in the draining lymph nodes (LNs). Despite the activation of tumorspecific T cells, the tumors grew progressively, indicating that the degree of immune activation induced by tumor growth was insufficient to ultimately eliminate the tumors. These results suggest that developing tumors can induce immune responses but may titrate their level of immune activation to one that ultimately does not ‘‘keep up’’ with tumor progression. Such a circumstance is one that is highly susceptible to the immune editing concept put forward by Scheriber and colleagues in which the tumor edits itself genetically to maintain a sufficient level of resistance to induced immune responses. In the case of the LCMV GP33 T antigen transgenic mice, because neither anergic nor deletional tolerance was observed, animals treated with the DC stimulatory anti-CD40 antibody demonstrated significant slowing of tumor growth. Thus, it may be possible under some circumstances to shift the balance between tumorimmune evasion and tumor-immune recognition by agents that affect the overall activation state of either antigen-presenting cells (APCs) or T cells (see later). It has been more difficult to obtain definitive evidence that human cancers tolerize tumor-specific T cells since humans cannot be manipulated the way mice are. However, the T cells that are grown out of patients with cancer tend to be either of low affinity for their cognate antigen or recognize antigens that bind poorly to their presenting HLA (human MHC) molecule, resulting in inefficient recognition by T cells. Recently, the first crystal structure of the TCR-peptide-MHC trimolecular complex has been solved for an MHC class II–restricted human tumor antigen (90). Interestingly, the orientation of the TCR, which is of low affinity for the peptide-MHC complex, is distinct from trimolecular complexes for viral (foreign) antigens and is partially similar to trimolecular complexes for a selfantigen. Thus, there may be fundamental structural features of tumor antigen recognition that lie between those of foreign antigen and self-antigen recognition. As will be discussed in the following text, one of the features of the tumor microenvironment that is likely central to the capability of tumors to tolerize tumor-specific T cells is the immature or inactive state of tumor-infiltrating DCs. DCs are the major APC that present peptides to T cells to initiate adaptive immune responses. In the context of infection, micro-

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Figure 2 Two pathways for DC activation. DCs, the major antigenpresenting cells that regulate T-cell activation, develop from hematopoietic precursors under the control of various cytokines, including GM-CSF. In the presence of activating signals from cytokines such as TNF or exogenous pathogen-associated ligands for toll-like receptors (such as LPS or unmethylated CpG sequences), DCs become activated to express high levels of MHC molecules, chemokines to attract T cells and costimulatory signals, including B7 family members, TNF family members, and proinflammatory cytokines (pathway on right). The result is activation of T cells specific for antigens presented by these DC. In the absence of DC activation signals, there is steady-state presentation of selfantigens by immature DC that do not provide adequate costimulatory signals for T-cell activation (pathway on left). The result is tolerance induction among T cells specific for self-antigens presented by these unactivated DC. Tumor-associated DC are not fully activated and thus can induce tolerance among tumor-specific T cells. Abbreviations: DC, dendritic cell; LPS, lipopolysaccharide.

bial ligands or endogenous ‘‘danger signals’’ associated with tissue destruction activate DCs to a state whereby they present antigens to T cells together with costimulatory signals that induce T-cell activation and development of effecter function. However, in the absence of microbial products or danger signals, DCs remain in an immature state in which they can still present antigens to T cells but without costimulatory signals. These immature DCs function as ‘‘toleragenic’’ DCs, inducing a state of antigen-specific T-cell unresponsiveness [termed anergy (Fig. 2)]. It is thought that steady-state presentation of self-antigens by immature DCs is an important mechanism of peripheral self-tolerance. Thus, if a tumor is able to produce factors that inhibit local DCs from becoming activated in response to the endogenous danger signals associated with tissue invasion, it could shift tumor-specific T cells from a state of activation to one of tumor-specific tolerance.

REGULATORY T CELLS AND CANCER Over the past 10 years, regulatory T cells (Treg) have emerged as a central player in maintenance of the tolerant state as well as general downregulation of immune responses to pathogens (91,92). Not surprisingly, they appear to play a role in tolerance to tumor antigens as well as the resistance of tumors to immune-mediated elimination (93,94). In contrast to the

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ephemeral CD8 suppressor cells of the 1970s that failed to withstand experimental scrutiny, the more recently defined CD4þ Treg are characterized by expression of a central master regulatory transcription factor—FoxP3—whose role in the gene expression programs of Treg is being actively studied (95). While CD4þ Treg selectively (but not specifically) express a number of cell membrane molecules, including CD25, neuropilin, glucocorticoid-induced tumor necrosis factor receptor (GITR), and lymphocyte activation gene-3 (LAG-3) (91,96–98), their overall genetic program and inhibitory capacity are absolutely dependent on sustained expression of Foxp3 (99,100). Mechanisms of immune suppression by Treg vary and include production of inhibitory cytokines such as IL-10 and transforming growth factor (TGF)-b (101–103). In keeping with the emerging appreciation that tumors are by nature highly tolerogenic, numerous murine studies have demonstrated that Treg expand in animals with cancer and significantly limit the potency of antitumor immune responses—either natural or vaccine induced. For example, in a study by Sutmuller and colleagues, a combination of GM-CSF-traduced tumor vaccine plus anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibodies was much more effective at eliminating established tumors when animals were treated with anti-IL-2 receptor a antibodies to eliminate CD4þ Treg (104). It is now appreciated that treatment with low-dose cytoxan is a relatively simple and reasonably effective way to temporarily eliminate cycling Treg (105–108). This appears to be a major mechanism by which pretreatment with low-dose cytoxan prior to vaccination can significantly enhance the capacity of vaccines to break tolerance. As new cell membrane molecules that define Treg are identified, the capacity to block regulatory T-cell activity with antibodies to these molecules presents new opportunities for immunotherapeutic strategies to break tolerance to tumor antigens.

ONCOGENIC PATHWAYS ACTIVELY MEDIATE TUMOR–IMMUNE SYSTEM INTERACTIONS The previous sections outline the complex interplay between tumor and host immune system and describe the experimental evidence that the immune system is in general tolerant to tumors and their antigens under circumstances in which a tumor has established and is expanding within the host. Is this tolerance to tumor antigens a passive default pathway or does the tumor actively manipulate its immune microenvironment in a way to render the immune system tolerant to its antigens? Indeed, evidence is accumulating that activation of oncogenic pathways in the tumor as well as inactivation of tumor suppressor genes have immunologic consequences far beyond the more commonly studied roles in growth regulation and anti-apoptosis. Critical signaling pathways whose role has been studied in this context include STAT3, NF-kB, Braf, and phosphatase and tensin homolog (PTEN). While each of these pathways (either activation or inactivation) has been well studied for its role in ‘‘classic’’ tumor biology such as dysregulated growth, regulation of apoptosis, and resistance to DNA damaging agents, additional roles in the organization of the immune microenvironment of the tumor have also been elucidated recently. The best-studied oncogenic pathway to play a role in tumor-immune evasion is the STAT3 pathway. STAT3 is one of two STATs (the other being STAT5A) to be constitutively activated in many diverse tumor types (109–112). Activation of STAT3 involves tyrosine phosphorylation resulting in homodimerization in the cytosol that leads to nuclear transport where it

participates in transcriptional activation (and, in some cases, repression) of diverse genes. Although synthetic mutations in STAT3 can confer on it oncogenic activity, constitutive activation of STAT3 in tumors is not a consequence of mutation. Instead, STAT3 is downstream of a number of important oncogenic tyrosine kinases, both receptor tyrosine kinases and src family tyrosine kinases. A number of receptor tyrosine kinases that play important roles in human cancer, including EGFR, HER2/Neu, and cMet, signal in part through STAT3 (113–115). In addition, src and potentially other src family tyrosine kinases can activate STAT3 (116). In fact, the original association of STAT3 with oncogenesis came from the demonstration that src-dependent transformation required STAT3 (117). Activated STAT3 in tumors participates in transcriptional activation of a number of genes associated with common cell autonomous and noncell autonomous mechanisms of carcinogenesis and cancer promotion. These include cell cycle regulation (e.g., cyclin D1), antiapoptosis (e.g., B cell lymphoma–X large (Bcl-Xl) and survivin) and angiogenesis (e.g., vascular endothelial growth factor (VEGF)) (118,119). In addition, STAT3 activation in tumors has been shown to repress the production of proinflammatory cytokines and chemokines that could enhance antitumor-immune responses (120). These include proinflammatory cytokines such as type I IFN and TNF as well as proinflammatory chemokines such as regulated upon activation, normal T-cell expressed, and secreted (RANTES) and IP-10. Thus, blockade of STAT3 signaling in tumor cells results in the release of multiple proinflammatory mediators and consequent infiltration with cells of both the innate and adaptive immune system that ultimately inhibit tumor growth. Beyond simply repressing the production and release of molecules that could promote antitumor-immune responses, STAT3 signaling also induces the release of factors that inhibit activation of multiple immune cell types in the tumor microenvironment. These include DCs, NK cells, and granulocytes, which, though present in significant numbers within tumors, are generally found in an unactivated state. Some of the STAT3 regulated factors that induce this ‘‘quiescent microenvironment’’ include IL-10, VEGF, IL-6, and possibly IL-23. As will be described in the following text, some of these cytokines promote distinct forms of immune responses that promote rather than inhibit tumor growth. The receptors for each of these factors are expressed on cells of the hematopoietic system, and they themselves signal through STAT3. Thus, infiltrating hematopoietic cells within the tumor microenvironment are found to also express constitutively activated STAT3. Blockade of STAT3 in the hematopoietic system (e.g., via hematopoietic specific STAT3 knockout) results in dramatically enhanced activation of DCs and cells in the innate immune system (such as NK cells and granulocytes) and leads to antitumor immune responses. In fact, even aggressive tumors fail to grow when transplanted into animals with hematopoietic STAT3 knockout (121). Thus, STAT3 appears to be an important global signaling pathway that restrains antitumor immunity. Another immunologically relevant pathway that is commonly constituently activated in cancer is the NF-kB pathway (122,123). Normally, NF-kB is activated in a highly stimulusdependent fashion but is constitutively activated in many types of tumors. Multiple NF-kB family members participate in either a canonical or noncanonical NF-kB activation pathway. Common to both pathways is the activation of IkB kinase (IKK), which phosphorylates IkB, leading to ubiquitin-dependent degradation and release of NF-kB to traffic from the cytosol

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to the nucleus and activate gene transcription programs (124). Alternatively, IKK phosphorylation can result in cleavage of a precursor protein for the activation of the noncanonical NF-kB pathway. The mechanism for constitutive NF-kB activation in tumors is not currently known. Normally, NF-kB plays a central role in the activation of virtually all cells in the immune system—both innate and adaptive. In the case of innate immunity, toll-like receptors (TLRs) on the surface of cells or intracellular sensors of viral RNA or DNA (the retinoic acid inducible gene I (RIGI) or melanoma differentiation-associated gene-5 (MDA-5) pathway) result in a signaling cascade that activated NF-kB via tumor necrosis factor receptor associated factor 6 (TRAF6) (125,126). Paradoxically, constitutive activation of NF-kB in tumors is associated predominately with activation of anti-apoptotic genes, whereas many of the typical NF-kB responsive proinflammatory/proimmunity genes are not activated in tumors. Recently, it was demonstrated that the selective NF-kB gene activation program in tumors is dependent on its association with STAT3. Indeed, coactivation of STAT3 and NF-kB is commonly observed in tumors. This coactivation appears, in part, to be due to a newly defined role for STAT3 in enhancing acetylation of NF-kB p50 subunit, resulting in enhanced retention of active NF-kB in the nucleus of tumor cells. This retention appears to be through the p300 acetyl transferase. The result is a shift in equilibrium toward nuclear retention of NF-kB. In addition, STAT3-NF-kB complexes fail to bind promoters of proinflammatory/proimmunity genes that are typically repressed in tumor cells, whereas STAT3 NF-kB dimers are found to be associated with promoters driving anti-apoptotic genes such BCL-Xl and surivin. These findings highlight the interactivity between key signaling pathways of tumor cells as well as the interplay between gene expression programs mediating tumor immunity verses tumor survival. An additional oncogenic pathway that appears to play a role in tumor immune evasion is the Braf pathway (127). Braf is constitutively activated in the majority of human melanomas because of a single activating mutation. Kawakami and colleagues have demonstrated that factors produced by melanoma cells that inhibit DC activation are in part driven by Braf. Knockdown of Braf with siRNA abrogates the production by melanoma cells of factors that inhibit DC activation. This inhibition appears to be independent of but complementary to that provided by STAT3 activation in melanoma cells. Thus, it appears that multiple oncogenic pathways active in tumor cells may contribute to the release of factors that inhibit DCs and other components of innate immunity, shifting the balance of immune responses toward tolerance. In addition to oncogenic pathways, inactivation of tumor suppressor pathways may also play a role in immune evasion by tumors. In one example, Pan and colleagues demonstrated that expression of a T cell–inhibitory molecule by tumors, B7-H1 (see the following text), is linked to inactivation of PTEN. PTEN, an inhibitor of the oncogenic AKT pathway, is emerging as one of the most important tumor suppressor pathways in cancer (128). More recently, Lowe and colleagues provided evidence that the p53 pathway may play a role in inhibiting innate immune responses to tumors. In a transgenic system in which inactivated p53 is conditionally reexpressed in tumors, they found that the inhibition of tumor growth induced by reactivation of p53 might be in part dependent on induction of innate immune responses mediated by NK cells (129). Taken together, these findings strongly suggest that oncogene and tumor suppressor gene pathways in tumors

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play important roles in orchestrating the interaction between the tumor cell and its immune microenvironment so that immune responses induced by the invasion and metastasis process do not eliminate the tumor cell itself. While most of the focus on the function of oncogenic and tumor suppressor pathways has been on cell autonomous functions within the tumor such as growth regulation, there is growing appreciation that these pathways additionally affect the tumor microenvironment via nontransformed cells. As an integral part of the tumor microenvironment, the immune system is clearly subject to regulation by these pathways. Understanding of the immunologic consequences of these pathways ultimately provides direct opportunities to develop therapeutic approaches that integrate inhibitors of oncogenic pathways, activators of tumor suppressor pathways, and other immunotherapeutic approaches to cancer.

IMMUNOLOGIC CHARACTERISTICS OF THE TUMOR MICROENVIRONMENT Ultimate understanding of the relationship between the tumor and the host immune system requires elucidation of local cross talk at the level of the tumor microenvironment. As mentioned at the outset, the hematopoietic/immune system is a major component of the tumor microenvironment. The systemic tolerance to tumor antigens begins with events that occur in this microenvironment. Beyond mechanisms that skew tumorspecific T cells toward immune tolerance, the tumor microenvironment is replete with mechanisms that dampen antitumorimmune responses locally (Fig. 3). This represents an important barrier to successful immunotherapy even when activated effector responses can be generated with vaccines. As the specific cells and molecules within the tumor microenvironment that mediate this hostile immune environment are elucidated, inhibitors are being developed and tested to use as adjuncts to vaccination that will allow activated immune cells to function more effectively within the tumor microenvironment. The previous section described how oncogenic pathways in the tumor cell directly affected the immune microenvironment of the tumor. In addition to its role in inhibiting the activation and effector function of DC, granulocytes, and NK cells in the tumor microenvironment, STAT3 signaling has also been reported to play a role in guiding immature myeloid cells (iMC) in the tumor microenvironment to differentiate into myeloid suppressor cells (MSC) rather than DC with APC activity. iMC (130,131) and MSC (132–135) represent a cadre of myeloid cell types, including tumor-associated macrophages (TAM), that share the common feature of inhibiting both the priming and effector function of tumor-reactive T cells. It is still not clear whether these myeloid cell types represent distinct lineages or different states of the same general immune inhibitory cell subset. In mice, iMC and MSC are characterized by coexpression of CD11b (considered a macrophage marker) and Gr1 (considered a granulocyte marker) while expressing low or no MHC class II or the CD86 costimulatory molecule. In humans, they are defined as CD33þ but lacking markers of mature macrophages, DCs, or granulocytes and are DR negative. A number of molecular species produced by tumors tend to drive iMC/MSC accumulation. These include IL-6, CSF-1, IL-10, and gangliosides. IL-6 and IL-10 are potent inducers of STAT3 signaling. Another cytokine reported to induce iMC/MSC accumulation is GM-CSF (136). This finding is somewhat paradoxical since GM-CSF is a critical inducer of DC differentiation, and

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Figure 3 The hostile immune microenvironment of the tumor. Activation of oncogenic pathways and inactivation of tumor suppressor pathways in the tumor lead to a cascade of molecular and cellular processes in the tumor microenviroment that block the killing function of innate immune effectors such as NK cells and granulocytes and block DC maturation (Figs. 3 and 4). In addition, multiple cell membrane molecules such as IL-10, transforming growth factor b, B7-H1, and B7-H4 are upregulated. These molecules bind to receptors that inhibit Tcell effector function. Immature myeloid cells (iMC) produce NO that inhibits T cells, and immature plasmacytoid DC (iPDC) produce indoleamine dioxygenase (IDO), which depletes tryptophan. Regulatory T cells also accumulate in the tumor microenvironment, further blunting antitumor T-cell responses. Abbreviation: DC, dendritic cell.

GM-CSF-transduced tumor vaccines enhance antitumor T-cell immunity via accumulation of DCs at the vaccine site followed by increased DC numbers in vaccine draining LN. It appears that the paradox is solved on the basis of levels of GM-CSF. High local levels drive DC differentiation at the vaccine site, whereas chronic production of low levels of GM-CSF can promote iMC/MSC accumulation. GM-CSF-transduced vaccines that produce extremely high GM-CSF levels can induce iMC/MSC accumulation at distant sites (i.e., spleen and LNs) because they release enough GM-CSF systemically to drive iMC/MSC accumulation. A number of mechanisms have been proposed to explain how iMC/MSC inhibit T-cell responses within the tumor microenvironment. Most include the production of reactive oxygen species (ROS) and or reactive nitrogen species (RNS). NO production by iMC/MSC as a result of arginase activity, which is high in these cells, has been well documented, and inhibition of this pathway with a number of drugs can mitigate the inhibitory effects of iMC/MSC. ROS, including H2O2, have been reported to block T-cell function associated with the downmodulation of the z chain of the TCR signaling complex (137), a phenomenon well recognized in T cells from cancer patients and associated with generalized T-cell unresponsiveness. Another mediator of T-cell unresponsiveness associated with cancer is the production of indoleamine 2,3-dioxygenase (IDO) (138). IDO appears to be produced by DCs either within tumors or in tumor-draining LN. Interestingly, IDO in DCs has been reported to be induced via backward signaling by B7-1/2 upon ligation with CTLA-4 (139,140). Apparently, the major IDO-producing DC subset is either a plasmacytoid DC (PDC)

or a PDC-related cell that is B220þ (141). IDO appears to inhibit T-cell responses through catabolism of tryptophan. Activated T cells are highly dependent on tryptophan and are therefore sensitive to tryptophan depletion. Thus, Munn and Mellor have proposed a bystander mechanism, whereby DCs in the local environment deplete tryptophan via IDO upregulation, thereby inducing metabolic apoptosis in locally activated T cells. Another inhibitory molecule produced by many cell types that has been implicated in blunting antitumor immune responses is TGF-b, which is produced by a variety of cell types, including tumor cells, and which has pleotropic physiologic effects. For most normal epithelial cells, TGF-b is a potent inhibitor of cell proliferation, causing cell cycle arrest in the G1 stage (142). In many cancer cells, however, mutations in the TGF-b pathway confer resistance to cell cycle inhibition, allowing uncontrolled proliferation. Additionally, in cancer cells, the production of TGF-b is increased and may contribute to invasion by promoting the activity of matrix metaloproteinases. In vivo, TGF-b directly stimulates angiogenesis; this stimulation can be blocked by anti-TGF-b antibodies (143). A bimodal role of TGF-b in cancer has been verified in a transgenic animal model using a keratinocyte-targeted overexpression (144). Initially, these animals are resistant to the development of earlystage or benign skin tumors. However, once tumors form, they progress rapidly to a more aggressive spindle cell phenotype. While this clear bimodal pattern of activity is more difficult to identify in a clinical setting, it should be noted that elevated serum TGF-b levels are associated with poor prognosis in a number of malignancies, including prostate cancer (145), lung cancer (146), gastric cancer (147), and bladder cancer (148).

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From an immunologic perspective, TGF-b possesses broadly immunosuppressive properties and TGF-b knockout mice develop widespread inflammatory pathology and corresponding accelerated mortality (149). Interestingly, a majority of these effects seem to be T cell mediated, as targeted disruption of T-cell TGF-b signaling also results a similar autoimmune phenotype (150). Recent experiments by Chen and colleagues rather convincingly demonstrated a role for TGF-b in Tregmediated suppression of CD8 T-cell antitumor responses (151). In these experiments, adoptive transfer of CD4þ CD25þ Treg inhibited an antitumor CD8 T-cell effector response, and this inhibition was ameliorated when the CD8 T cells came from animals with a dominant negative TGF-b1 receptor. One of the unresolved issues in the study of tumor immune evasion relates to the mechanisms by which tumors induce antigen-specific T-cell tolerance. While the many mechanisms described in the preceding text, including STAT3 signaling–dependent mechanisms, IDO, ROS, RNS, TGF-b, etc., clearly inhibit priming of T-cell responses and/or tumor killing by activated effector T cells, it remains to be definitively determined which processes actively induce antigen-specific T-cell tolerance that has been documented in transgenic models. Self-tolerance induction for peripheral tissue antigens is now thought to involve specific presentation of tissue-specific antigens to mature T cells in the absence of appropriate costimulatory signals. Similar mechanisms are likely operative in the case of tumor-induced tolerance. Originally, the relevant costimulatory signals were envisioned to be provided by B7 family costimulatory molecules expressed by DCs (152). It is now becoming clear that additional proinflammatory cytokines such as IFN, IL-12, TNF, etc., are critical in the distinction between effector T-cell induction and tolerance induction. An emerging concept is that immature or not fully mature DCs are critical in presenting self-antigens to induce T-cell tolerance in the absence of TLR-mediated danger signals associated with infection (153,154). Unquestionably, DCs found within the tumor microenvironment have a relatively immature, unactivated phenotype characterized by low levels of proinflammatory cytokine production and CD86 and surface MHC class II expression. As described earlier, a major inhibitory signaling pathway induced in tumor-infiltrating DC is the STAT3 pathway which, when activated, strongly antagonizes TLR- and CD40-mediated DC activation. As mentioned, tumorderived factors such as IL-10, IL-6, and VEGF (in part induced by STAT3 signaling in the tumor cell) can induce STAT3 activation in DCs. As described in the previous section, constitutive V-raf murine sarcoma viral oncogene homolog B1 (BRAF), signaling in melanoma cells has additionally been shown to induce release of factors that inhibit DC activation (127). These immature ‘‘activation-inhibited’’ DCs clearly represent a prime candidate for the induction of tumor-specific T-cell tolerance. It remains an open question as to whether iMC/MSC represent a distinct intertumoral cell subset capable of presenting antigens to T cells in a toleragenic fashion (155). A recent report indeed suggested that iMC loaded with antigen and adoptively transferred into mice can induce antigen-specific T-cell tolerance. Finally, it has been suggested that IDOexpressing DC can induce antigen-specific T-cell tolerance because IDO-mediated tryptophan selectively kills or inhibits proliferation of activated T cells (156). According to this model, IDO-expressing DCs would present antigen to T cells inducing activation followed by activation-associated cell death mediated

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by depletion of local tryptophan stores by the IDO in the presenting DCs. As described in the following text, Treg play an additional important role in induction of or maintenance of tumor antigen-specific T-cell tolerance. Whether Treg mediate T-cell tolerance independently from immature or toleragenic APCs or whether the two mechanisms are completely interrelated (i.e., toleragenic DCs inducing a Treg phenotype among antigen-specific T cells and antigen-specific Treg acting on DCs to enhance their toleragenic capacity) remains to be definitely determined.

DENDRITIC CELLS: THE KEY TARGET OF CANCER VACCINES The central theme among cancer vaccination strategies is enhancement of modulation of APC function. This is based on the concept that the quantitative and qualitative characteristics of T-cell responses to antigen depend on the signals they receive from the APC. Among the major bone marrow–derived APC subtypes (B cells, macrophages, and DCs), the DC has emerged as the most potent APC type responsible for initiating immune responses (157,158). As described earlier, DCs associated with cancer have altered properties that result in failure to activate T cells optimally. Cancer vaccines in essence seek to skew the function of DCs toward generation of effector T-cell responses. As virtually all phases of DC differentiation and function can be modulated by engineered vaccines, it is important to understand the molecular signals that regulate their role in activation of T cell–dependent immunity (Fig. 2). At sites of infection and inflammation, bone marrow–derived progenitor cells respond to both proliferative and differentiation signals. GM-CSF, as well as other cytokines such as Fms-like tyrosine kinase-3 ligand (FLT-3L) and IL-4, serve as mitogenic or comitogenic factors that induce an intermediate stage of DC differentiation, characterized by efficient antigen uptake and processing (159–163). Once they have ingested antigens at inflammatory sites in the tissue, immature DCs differentiate in response to a number of distinct ‘‘maturation’’ signals. While many diverse molecules induce DC maturation, most appear to signal DCs via binding to two classes of receptor—TLRs and the TNF receptor (TNFR) family. TLRs are ‘‘pattern recognition receptors’’ (PRR), which bind common chemical moieties expressed by pathogens termed pathogen-associated molecular patterns (PAMP) such as lipopolysaccharide (LPS) and unmethylated CpG DNA sequences (164). The two best-characterized endogenous DC maturation factors are TNF-a itself and CD40L (165–167). In addition to TLRs, intracellular PRR, including protein kinase R (PKR), RIGI, MDA-5, and NOD1/2, recognize PAMP from intracellular bacteria and viruses that invade the cytosol (126,168,169). Maturation of DCs, which occurs as they traffic to draining LNs, is characterized by transport of peptide-MHC complexes to the cell surface (170,171). In addition to provision of high densities of peptide-MHC complexes for T-cell stimulation (termed signal 1), DCs regulate T-cell activation and differentiation through provision of costimulatory signals in the form of cytokines, such as IL-12, and membrane-bound ligands of the B7 and TNF family (collectively termed signal 2). The everexpanding panoply of costimulatory signals utilized by DCs to instruct T cells as to their pathway of differentiation and effector function defines a high degree of complexity to the communications that occur between APC and T cells. When immature DCs present antigens to T cells in the absence of

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costimulatory signals, the outcome is tolerance induction. This is a normal mechanism for maintenance of tolerance to selfantigens. It is also a mechanism by which tumors can induce immune tolerance to their own antigens (Fig. 2). As discussed throughout this chapter, tumor-induced immune tolerance is a major barrier to successful vaccination of established cancers. Each of the molecular events involved in proliferation, antigen presentation, and costimulation represents potential targets that are being exploited in the design of immunotherapy approaches.

WHOLE-CELL TUMOR VACCINES Overview Prior to the molecular identification of tumor-specific antigens, investigators used tumor cells themselves as a source of tumor antigen (Table 2). Efforts to modify tumor cells as vaccines date back roughly half a century. Whole-cell tumor vaccines have been generated through mixing with adjuvants aimed at enhancing ‘‘immunogenicity’’ of tumor-specific or tumorselective antigens incorporated therein, with clinical testing of these mixtures dating back to the 1980s (172–176). Another approach has been to hapten modify whole tumor cells with chemicals such as dinitrophenol (177) or infect them with a virus (178). The general concept is that increasing the immunogenicity of tumor cells using either adjuvants or expression of foreign antigens will enhance immune responses to the endogenous tumor antigens, thereby allowing the immune system to kill metastatic tumor deposits. More recently, a new era in genetically engineered whole-cell vaccination has involved the modification of tumor cells through transfer of genes encoding cell membrane immunostimulatory molecules or cytokines. While most of the clinical activity related to adjuvanted whole-cell vaccines is diminishing significantly, active clinical investigation continues for cytokine gene–modified whole-cell vaccines, particularly with the GM-CSF gene (described later). Adjuvanted wholecell tumor vaccines have been tested extensively in patients with melanoma, renal cell carcinoma, and colorectal carcinoma. Most of these vaccine strategies have involved coinjection of either autologous or allogeneic tumor cells with adjuvants such as Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum (172–176,179). While BCG and C. parvum were long known to

represent reasonable vaccine adjuvants for generation of antibody responses, a limitation of this vaccination approach has been their relatively poor capacity to generate T-cell responses, particularly in the face of established tolerance. Initially, nonrandomized clinical trials were performed, which demonstrated hints of promise. In some of these studies that reported antitumor responses, the responses were shown to correlate with the return of delayed type hypersensitivity (DTH) responses to recall antigens and, more importantly, with the development DTH responses to autologous tumor cells. Application of BCG-adjuvanted tumor cell vaccines to patients with bulky metastatic cancer demonstrated an insignificant clinical response rate. However, given the plethora of studies in animal models suggesting that cancer vaccination might be more effective in the setting of minimal residual disease, a number of studies employing BCG-adjuvanted tumor vaccines clinical trials were undertaken in the minimal residual disease setting postresection of the primary tumor. Initial enthusiasm for a BCG-adjuvanted, autologous colon cancer vaccine in patients with resected stage 2/3 colon cancer (180) as well as a melanoma vaccine consisting of a mixture of irradiated allogeneic human melanoma lines with BCG utilized in melanoma patients with stage 3 and resected stage 4 disease (181) was based on phase 2 studies and limited singleinstitution phase 3 studies. The concern in the interpretation of clinical outcomes of these phase 2 studies is that it was unclear whether the untreated ‘‘historical controls’’ were truly comparable to the population of patients treated in the phase 2 studies. In the absence of careful case-controlled comparisons, ultimate acceptance of these vaccines depended on pivotal randomized phase 3 studies in which both progression-free survival and overall survival were the relevant clinical end points. In the case of the autologous BCG-adjuvanted colon cancer vaccine, an initial randomized single-institution study in the Netherlands claimed a longer overall survival in patients with stage 2 but not stage 3 colon cancer (182). Unfortunately, these findings were not reproduced in expanded multi-center trials, possibly owing, in part, to technical difficulties in consistent autologous tumor preparation as part of the patientspecific vaccine formulation (183). After 20 years of phase 1 and 2 studies with an allogeneic BCG-adjuvanted melanoma vaccine, a randomized phase 3 clinical trial between BCGadjuvanted allogeneic melanoma cells versus BCG control

Table 2 General Categories of Antigen-Specific Cancer Vaccine in Clinical Testing Vaccine type

Advantages

Disadvantages

Peptide

l

l

Easy to produce

l l

Protein DNA

l

l l

Virus

l l

Easy to produce

l l

Poor immunogenicity Poor cytotoxic T lymphocyte induction

Easy to produce Versatile construction

l

Poor immunogenicity in humans

Good immunogenicity Versatile construction (some)

l

Safety (some viruses) Neutralizing immunity precludes revaccination Challenge to produce

l l

Bacterium

l l l l l

Poor immunogenicity HLA allele specific Can induce tolerance

Excellent immunogenicity Versatile construction Safety—antibiotic sensitive Repetitive vaccination Easy to produce

l l

Regulatory hurdles Potential toxicity from bacterial products

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demonstrated no evidence of enhanced overall survival for the BCG plus tumor vaccine arm (184). While the phase 2 studies claimed to have demonstrated significant survival benefit relative to case-matched controls, the case-matched controls demonstrated suspiciously short overall survival times relative to melanoma patients of similar stage from multiple other clinical studies. There were encouraging reports of responses to vaccination with BCG-adjuvanted dinitrophenol (DNP)-modified allogeneic melanoma vaccines (185). However, definitive randomized phase 3 trials have not been completed at the time of this writing. While a number of these studies reported that patients with enhanced DTH responses postvaccination had better disease outcomes than patients who did not, these studies were largely devoid of analyses of antigen-specific T-cell responses, and it is unclear whether the association between DTH responses and enhanced survival had anything to do with the vaccination. A similar fate befell the melanoma vaccine Melacine, a mixture of lysates from multiple allogeneic melanoma cells admixed with the ‘‘detoxified’’ lipopolysaccharide derivative monophosphoryl lipid (MPL) A plus mycobacterial cell wall extracts. Despite encouraging reports from phase 2 studies, a definitive phase 3 study in patients with stage 2/3– operated melanoma failed to demonstrate a statistically significant effect on overall survival (186). A retrospective subset analysis suggested that HLA-A2þ and HLA-C1þ patients had greater benefit, but this result has not been confirmed in a prospective trial. One of the limitations of these trials is that none demonstrates definitive enhancement of T-cell responses against relevant antigens. In the case of melanoma, many tumor antigens recognizable by T cells are indeed well defined and responses to them should be measured as part of the development process. As described earlier, a more limited set of ‘‘immunorelevant’’ antigens are defined for other human cancers. In summary, the age of adjuvanted whole-cell or lysate tumor vaccines appears to be slowly drawing to a close and will likely be a historical footnote in the development of cancer immunotherapies.

GENETICALLY MODIFIED TUMOR VACCINES With the development of improved genetic techniques, emphasis shifted to genetic modification of tumor cells to express immunostimulatory molecules. Building on the original studies of Lindenman and Klein (187), who showed that vaccination with influenza virus–infected tumor cell lysates generated enhanced systemic immune responses against challenge with the original wild-type tumor, Fearon and colleagues use direct gene transfer to introduce the immunogenic influenza hemagglutinin (HA) gene into murine tumor cells to create genetically engineered vaccines (188). These HA transfectants induced a systemic immune response against challenge with the parental tumor. Gene transfer of viral antigens was eventually superceded with gene transfer of immune response–modulating genes. It is important to point out that although many of the strategies were designed with a specific mechanism in mind, it is becoming clear that genetic manipulation to alter expression of even a single gene product can result in a complex cascade of cellular responses in vivo that ultimately may affect multiple aspects of antigen processing, presentation, and costimulation. There are many ways to genetically modify tumor cells to augment T cell–mediated antitumor immunity. One involves the genetic modification of tumor cells to express cytokines that

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function as attractants or differentiating agents for dedicated APCs such as DCs (189). Recruited DCs ingest released tumor antigens at the site of vaccination and present them together with appropriate costimulation required for the activation of a tumor-specific T-cell response. Alternatively, the tumor cell can be genetically modified so that it becomes the APC itself. Both ex vivo and in vivo methods of gene delivery have been employed in the development of genetically modified whole-cell cancer vaccines. Ex vivo gene delivery involves the modification of cultured cells. The genetically modified cells are subsequently administered to the host, typically after irradiation. Clearly, the most effective way to enhance expression of MHC molecules or to enhance expression of costimulatory molecules such as B7.1 or B7.2 is to genetically modify the tumor cell itself. However, when the goal is to deliver cytokines locally in a paracrine fashion, genetic modification of the tumor cells themselves is not necessary. A number of transduced bystander cytokine delivery systems have been developed (190). The efficacy of bystander cytokine delivery systems is comparable to that of direct gene modification of the tumor cell for augmenting antitumor immunity. It is, however, necessary that the transduced bystander cells are admixed with the tumor cells in an appropriate ratio. Genes that encode cytokines are the most common types of genes that have been introduced into tumor cells to generate genetically modified tumor vaccines (189). Tumor cells transduced with cytokine genes alter the local immunologic environment at the vaccine site, enhancing either the presentation of tumor-specific antigens to the immune system or the activation of tumor-specific lymphocytes. Critically, the cytokine is produced at very high concentrations in the vicinity of the tumor, whereas systemic concentrations are relatively low. This paracrine physiology much more closely mimics the natural biology of cytokine action than does the systemic administration of recombinant cytokines. Since the initial reports of enhanced antitumor responses after vaccination with IL-2-transduced tumor vaccines (191,192), many cytokine genes have been introduced into tumor cells with various effects on both tumorigenicity and immunogenicity. Some of these cytokines induce a local inflammatory response that results in elimination of the injected tumor. This local inflammatory response is most predominately dependent on components of innate immunity rather than the classic T cells. Ultimately, however, the most important outcome of vaccination is the generation of enhanced T-cell responses specific for the antigens expressed by the vaccinating tumor.

GM-CSF Gene–Transduced Tumor Vaccines Among the vast array of cytokine gene–transduced tumor vaccine studies, GM-CSF-transduced tumor vaccines remain as the most actively pursued clinically despite the recent failure in phase 3 trials in prostate cancer. In the original study that identified GM-CSF, multiple cytokine, adhesion molecule, and costimulatory genes were introduced into the poorly immunogenic B16-F10 tumor using a replication defective retroviral vector that produced consistent high levels of expression of each of the transgenes in the absence of selection, thereby eliminating variability caused by different levels of gene expression and resultant cytokine expression. Animals were vaccinated with the irradiated transductants, followed by challenge with unirradiated wild-type B16-F10 cells to doses 3 to 4 logs higher than the minimal tumoricidal dose (193). Although a number of

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cytokine genes in that study, such as IL-4 and IL-6, induced some measurable systemic antitumor immunity (194,195), the most potent systemic antitumor effect was produced by GM-CSFtransduced tumor cells. Many subsequent studies in other murine tumor models have validated the potent systemic immunity induced by GM-CSF-transduced tumor vaccines. Antitumor immunity induced by GM-CSF-transduced vaccines has been shown to depend on CD4þ and CD8þ T cells. In addition to the classic MHC class I–restricted CTL, other effector arms mediated by CD4 cells have been shown to participate in the generation of maximal antitumor immunity. Th1 and Th2 effector arms have been delineated (196). The Th1 effector arm depends on g-IFN and involves the activation of macrophages at sites of metastases to produce reactive nitrogen species (NO), as well as reactive oxygen species (super oxides). Eosinophils appear to be important Th2 effectors that are dependent on the production of cytokines such as IL-4 and IL-5 by tumor-specific CD4 cells. The presence of eosinophils at DTH sites and in tumor metastases subsequent to vaccination with GM-CSF-transduced tumors is not only observed in animal models but has also been a consistent observation in clinical trials with different tumor types (197). Clinically, non-patient-specific GVAX platforms that use GM-CSF-modified allogeneic tumor cells exclusively offer numerous advantages over their autologous cell counterparts including manufacturing issues, consistency of vaccine, and limitation on vaccine quantity. Scientific data from both preclinical and clinical studies have provided support for the relevance of allogeneic GVAX immunotherapies and the seminal role of cross-presentation of allogeneic tumor–associated antigens by host APCs in the initiation of a cellular antitumor-immune response. The one drawback of allogeneic GVAX vaccines is that unique, patient-specific tumor antigens are not targeted.

ANTIGEN-SPECIFIC VACCINES The ultimate goal of cancer vaccine development is the use of antigen-specific vaccines that incorporate select tumor antigens into a vaccine vector(s) or adjuvanted formulation (Table 2). Antigen-specific vaccines have two intrinsic advantages over any type of cell-based vaccine. First, their formulation into a vaccine is much more versatile. Second, they do not contain the thousands of irrelevant or autoantigens included in a cell-based vaccines. The control over antigenic makeup afforded by antigen-specific vaccines is significant but requires knowledge of the best antigens to incorporate. The characteristics of an ideal tumor antigen include (i) highly selective expression by the tumor relative to normal tissues and expressed at reasonably high density on the surface of the tumor cell (as peptide-MHC complexes for T-cell recognition); (ii) shared among the majority of tumors of a particular type or, ideally, tumors with diverse histologies; (iii) provides a growth advantage for the tumor, ideally required for tumor growth or survival; and (iv) reasonable T-cell repertoire available in patients that has not been deleted and is not stringently tolerized. One of the major limitations in many of the antigen-specific vaccines tested clinically has been the application of antigens that do not meet these criteria. No one antigen may exist that does perfectly meet all these criteria. However, when planning to test a vaccine clinically, it is important to ask how well the candidate antigen(s) meets them. Ideally, antigen-specific vaccines should contain multiple immunorelevant antigens, particularly if they are not absolutely essential for tumor growth or survival. A final general principle is that a vaccine containing the best

tumor antigen(s) will not enhance antigen-specific responses effectively if the vaccine vector or adjuvant is suboptimal. These principles will be evaluated in the discussion below of commonly studies antigen-specific vaccines.

Peptide Vaccines The identification of T cell–recognized tumor antigens at the peptide level spawned a major effort beginning in the 1990s to develop peptide vaccines (198–200). The fundamental concept of peptide vaccination is that minimal peptides—particularly MHC class I–restricted peptides that are recognized by CD8 killer cells—can efficiently load MHC molecules on the surface of cells without requiring internal antigen processing routes. Early studies with peptide vaccines mixed with various adjuvants demonstrated induction of peptide-specific T cells in vivo and in some cases antitumor responses (201). Clearly, one of the major advantages of peptide vaccines is that they represent the ultimate defined tumor antigen, and therefore, the capacity to monitor induction of T-cell responses to the immunizing peptide is optimal. However, there are a number of disadvantages associated with peptide vaccination. First, individual peptides are selective for specific MHC alleles and therefore cannot be utilized generically. This limitation has been circumvented through the use of mixtures of peptides that bind to common MHC alleles, thereby assuring that the vast majority of patients will express at least one MHC allele that can present the peptide in the vaccine mix. Another major issue with minimal peptides as vaccinating antigens is that they do not only load the MHC molecules of DCs that would activate immune responses but will also bind to MHC molecules on the surface of cells other than DCs just as efficiently. The consequences of peptide presentation by these cells can be tolerance induction because they do not supply the appropriate costimulatory signals necessary for T-cell activation (202–204). Therefore, it is possible that peptide vaccination could, in fact, be detrimental for immune responses. Indeed, Melief and colleagues have presented evidence in animal models that vaccination with long peptides that require processing is significantly superior to vaccination with minimal MHC binding peptides (205). They have demonstrated that the advantage of vaccination with long peptides comes from the fact that only DCs can process long antigen-presenting peptides, thus leading to selective antigen presentation by DCs over other APCs that could induce tolerance. Another major factor in peptide vaccinations is the adjuvant that is used. Peptides themselves are intrinsically nonimmunogenic and strong immunization with peptides in animal models is only observed when strong adjuvants capable of activating DCs are mixed with the peptides. Peptides can also be loaded onto DCs grown ex vivo (see later) and reinjected into the patient. The most common formulation used for peptide vaccines in clinical trials is incomplete Freund’s adjuvant (IFA), an oil emulsion that does not contain any specific activators of DCs and is thus suboptimal. A few groups have reported enhanced immunogenicity of peptides conjugated to lipids (lipopeptide vaccines) (206,207). Clinical trials with peptide vaccines in cancer have predominantly utilized HLA class I–restricted tumor antigen peptides, but some are including MHC class II–restricted peptides (208). The inclusion of MHC class II–restricted peptides can either involve those derived from tumor antigens (such as tyrosinase in the case of melanoma) or involve peptides derived

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from foreign antigens that would nonspecifically stimulate CD4 helper cells that theoretically would provide help for enhanced stimulation of tumor-specific CD8 T cells responding to the tumor-specific MHC class I–restricted peptides. Clinical trials have been performed using peptide vaccines for many different cancer types, though vaccination for melanoma is the most common clinical target of peptide vaccines. A number of clinical trials using peptides either in the setting of bulky metastatic cancer or in the minimal residual disease setting have demonstrated induction of increased numbers of antigen-specific T cells using various methods with anecdotal clinical responses (209,210). Some methods utilize staining with peptide-MHC tetramers to directly visualize antigen-specific T cells. Other methods such as enzyme-linked immunosorbent spot (ELISPOT) or intercellular cytokine staining (ICS) seek to measure induction of functional T cells through the productions of cytokines such as g-IFN. Among the most interesting clinical results highlighting the dichotomy between induction of expanded numbers of peptide-specific T cells and the absence of clinical activity are the vaccine trials in melanoma that have utilized an anchormodified gp100 peptide to generate enhanced binding to HLAA2 (211). These trials utilize repetitive vaccination with this peptide in IFA in patients with no evaluable disease (NED) after resection for stage 2 to 4 melanoma. In these trials, Rosenberg and colleagues demonstrated the capacity to induce tremendous expansion of antigen-specific CD8 T cells, in some patients reaching 50% of the total circulating CD8 T cells as measured by staining with peptide-MHC tetramers and peptide-induced g-IFN production. Nonetheless, there was no evidence that relapse rate was significantly different from the relapse rate in the same group of melanoma patients not receiving vaccination. In some cases, relapsed melanomas could be demonstrated to have lost HLA-A2 expression or expression of the gp100 antigen, possibly representing an example of evasion or escape from the T-cell responses induced by vaccination. However, many of the relapsed tumors expressed HLA-A2 and gp100, thereby suggesting that the expanded populations of HLA-A2/gp100 specific T cells induced by peptide vaccination were ineffective at eliminating relapsing tumors. Rosenberg and colleagues recently summarized the clinical experience with peptide vaccines, indicating that peptide vaccines as single agents in the advanced disease setting provide a meager 2% to 3% objective response rate (212). However, more recent clinical studies, adding various adjuvants to peptide vaccines, mixing multiple MHC class I– and MHC class II–restricted tumor peptides together (213), and using ‘‘long peptides’’ (214), suggest that maximal potential benefits of peptide vaccines have yet to be realized. As more is learned about regulation of T-cell responses, it is quite plausible to imagine that T cells expanded in suboptimal conditions (i.e., in the absence of appropriate proinflammatory or costimulatory signals) could upregulate expression of inhibitory molecules that would block them from developing the critical effector activity necessary to kill tumor cells. Thus, tumor antigenspecific cells could expand but not be effective against tumors.

Ex Vivo Antigen-Loaded Dendritic Cell Vaccines The ability to culture DCs ex vivo has led to a plethora of studies of ex vivo antigen-loaded DCs as tumor vaccines. While DCs can be loaded with lysates of tumor cells, they are typically loaded with either peptides, recombinant protein or transduced

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with various vectors or RNA encoding specific antigens. Initially, it was demonstrated that loading of ex vivo cultured DCs with either MHC class I–restricted peptides, whole proteins or tumor lysates followed by administration back into the animal led to the generation of immune responses against the loaded antigen as well as antitumor responses (215–221). More recently, the advent of more efficient gene transfer vectors has led to approaches in which ex vivo cultured DCs are transduced with genes encoding relevant viral or tumor antigens (222–224). A number of recombinant replication defective viruses have been used to transduce DCs. In addition, Gilboa and colleagues have demonstrated that purified RNA can be used to effectively transduce DCs with resultant presentation of encoded antigens (225). This strategy offers the interesting possibility that DCs could be transduced with the entire amplified transcriptome of a tumor cell, even when only tiny amounts of tumor tissue is available. At present, the paucity of direct comparative studies, leaves open the question of which method of loading DCs ex vivo will be the most effective. Another major issue with ex vivo loaded DC vaccines is the degree of maturation that is induced in vitro and its relevance to homing and function of loaded DCs after reinjection. Maturation protocols utilized for DC vaccination are currently quite variable and range from monocyte conditioned medium to various defined agents such as TNF-a, IL-1, soluble CD40L, and prostaglandins (226,227). Concern has been raised that full-blown maturation/activation of DCs ex vivo to a stage normally achieved once they are within paracortical regions of the LN will impair their ability to home to LNs after reinjection. This has led to the suggestion that DCs should be loaded and reinjected in an immature state and allowed to mature in vivo. But such an approach has potential negative consequences as Steinman and colleagues have demonstrated—immunization of patients with antigenloaded immature DCs can actually result in tolerance or suppression of antigen-specific responses (228). Elucidation of proliferative and maturation signals for DCs has led recently to approaches in which DCs are not only loaded with antigen but are also transduced with genes encoding proliferation and maturation signals. This would result in autocrine DC stimulation in vivo after reinjection. In one study, DCs loaded with antigen were transduced with genes encoding GM-CSF and CD40L. These genetically modified DCs resulted in much more potent stimulation of antitumor immunity than immunization with DCs loaded with antigen alone (229). Another approach aimed at providing DCs with a full complement of tumor antigens is the generation of DC-tumor fusion vaccines (230,231). The concept behind this approach is to fuse autologous tumor cells with DCs, thereby allowing for the coexpression of all relevant tumor antigens together with all relevant DC molecules within the same cell. One of the major limitations to clinically translating an approach of this type is the efficiency with which fusion can be achieved between DCs and tumor cells in the absence of selection. Ultimately, it is critical that both preclinical and clinical DC vaccine studies identify the critical parameters of DC growth and maturation as well as antigen loading that result in therapeutically relevant levels of T-cell activation in vivo. Many clinical trials with DC vaccines have been performed using DCs cultured and activated in vitro by various methods and loaded with tumor antigens of various types. As with most cancer vaccines, melanoma is the most common target, though other cancers have been targeted as well. Induction of T-cell responses are commonly reported, and interesting

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anecdotal clinical responses have been reported in phase 1/2 trials (232). One of the more interesting clinical DC vaccine approaches involves transduction of DCs with RNA encoding telomerase that is targeted to the MHC class II processing pathway with the lysosome associated membrane protein (LAMP) targeting signal (233). Telomerase is the enzyme that restores telomeres, the ends of chromosomes. Without telomerase, cells will eventually stop growing when their telomeres are exhausted. Tumors typically upregulate telomerase, making it a tumor-selective antigen. Vaccination with DCs transduced with telomerase-LAMP RNA led to significantly enhanced CD4 and CD8 responses specific for telomerase. Two phase 3 clinical trials using DC vaccines have been negative, though one led to interesting politically motivated deliberations at the U.S. FDA. Schuler and colleagues compared DTIC chemotherapy with peptide-loaded DC vaccination in stage 4 melanoma patients (234). Objective responses were low in both arms (<5%), and there was no statistically significant difference in overall survival. A retrospective subset analysis suggested that HLA-A2þ/HLA-B44þ-patients might derive greater benefit from vaccination, but this has not been verified in a prospective manner. The Dendreon Corporation has recently reported results of a phase 3 trial in patients with advanced prostate cancer comparing placebo with a DC vaccine prepared by crude enrichment of peripheral blood lymphocyte (PBL) followed by culture with a prostatic acid phosphatase-GM-CSF fusion protein (235). The primary end point of prolonged progression-free survival was not achieved, but continued evaluation of the patients demonstrated prolonged overall survival compared with placebo of 4.5 months. The quality of this trial was questionable since the small size precluded careful matching of patient characteristics and registration was applied for on the basis of an end point different than that built into the original trial. In addition, initial evaluation of a follow-up phase 3 trial did not demonstrate even a significant trend toward improved overall survival in the DCvaccinated group. Although a majority of the advisory panel voted to approve the vaccine, the FDA ultimately chose to require additional supporting clinical data prior to approval (236). However, a follow-up phase 3 trial with overall survival as end point has recently been reported to have met its end point (see later) and, at this writing, is in the process of biologic license application (BLA) filing.

HEAT SHOCK PROTEIN–BASED VACCINES Another interesting category of proteins that may target antigen effectively to DCs and furthermore into MHC processing pathways, are the heat shock proteins (HSPs). It is now well established that complexing peptide antigens to certain HSPs such as gp96, hsp70, calreticulin, and hsp-110 enhances their immunogenicity significantly (237–244). HSPs were first utilized as tumor vaccines by purifying them from tumor cells followed by immunization. HSPs isolated from tumors are naturally complexed with a whole array of tumor-associated peptides. Other approaches to link antigen to HSP have included the production of recombinant fusion proteins in which antigenic peptides are covalently or noncovalently linked to the HSP (245) as well as DNA-based vaccines in which fusion genes between antigen and HSP gene are incorporated. In one direct comparative study using the human papillomavirus (HPV)-E7 antigen as a model, it was demonstrated that DNA vaccines encoding an E7-hsp70 fusion gene were 30-fold more

effective than the wild-type E7 gene in generating CD8þ responses (246). Immunogenic HSPs complexed with antigenic peptides have been shown to efficiently load the MHC class I processing pathway (so-called in vitro cross-presentation) (247). Although the intracellular pathway by which heat shock proteins effectively load MHC class I molecules with their associated peptides has not yet been elucidated, Srivastava and colleagues have identified CD91, the a2 macroglobulin receptor, as an important receptor for several heat shock protein (gp96, hsp70, hsp90) (248). Ultimately, the immunogenicity of HSPs has been proposed to result from their ability to activate APC and target antigens to MHC processing pathways. One report has suggested that hsp70 can activate macrophages via CD14/TLR-4 (LPS receptor)-dependent and CD14/TLR-4independent pathways (249). HSPs have also been reported to activate DCs (250) although the receptors that mediate these putative activation functions have yet to be elucidated. Clinical trials with heat shock proteins have been ongoing. A phase 2 trial vaccinating women with premalignant high-grade cervical dysplasia caused by HPV-16 using a bacterial hsp65-HPV16 E7 fusion protein demonstrated a 35% CR with induction of E7-specific T-cell responses in roughly half of the patients; however, the cohort was too small to determine whether this response rate was statistically different from the roughly 25% spontaneous regression rate observed in this patient group without treatment (251). The only phase 3 trial with heat shock protein vaccines reported to date was in patients with operated stage 2/3 renal cancer, who were randomized to observation or treatment with autologous hsp96 purified from the resected primary tumor. This was a negative study in that no statistical difference was observed in either relapse-free survival or overall survival. A second phase 3 trial of autologous hsp96 versus physician’s choice (IL-2, resection or chemotherapy) demonstrated no benefit relative to the physician’s choice arm but found that patients receiving 10 or more vaccine administrations had a longer overall survival than those who received lower numbers of vaccines (252).

THE GROWING ARMAMENTARIUM OF VACCINE VECTORS For all of the added value that recombinant DNA technology provides in engineering elements into vaccine constructs that enhance their potency, nature itself provides a virtually limitless array of delivery systems in the form of diverse microbes with potent intrinsic immunologic properties. These immunogenic properties derive from their expression of PAMP, which activate DCs via TLRs and intracellular sensing pathways such as PKR, RIGI, and MDA-5, their ability to induce proinflammatory cytokine expression by infected cells, and their ability to target intracellular MHC processing compartments. Of the three major microbial classes (viruses, bacteria, and fungi), viruses and bacteria have been the most intensively investigated. A few reports of engineered yeast vaccines emphasize the potential immunologic utility of the third microbial class.

Engineered Viruses Viruses are the most diverse and efficient gene transfer agents whose natural cell tropism and biologic features can significantly enhance the immunogenicity of antigens carried within them (Table 1). Using standard recombination approaches, Moss and Paoletti were the first to explore recombinant viruses

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as vaccine vectors. They utilized vaccinia virus, a highly immunogenic virus related to smallpox that is relatively nonvirulent in immunocompetent individuals. In most cases, a single immunization with recombinant vaccinia carrying a gene expressing an antigen will generate significantly greater immune responses against that antigen than the corresponding protein or peptide epitopes mixed with standard adjuvants (253–255). This is particularly true for CTL generation. To date, many viruses have been explored as recombinant vaccine vectors, including attenuated replication-deficient poxviruses (such as modified vaccinia ankara, fowlpox, and canarypox), Adenovirus, herpesviruses, and Venezuelan equine encephalitis virus (256–259). Each of these viruses has various advantages and disadvantages, and no clear ‘‘winner’’ has emerged as the absolute vector of choice. Features of viruses that can enhance their potency as vaccine vectors include their ability to induce immunologic ‘‘danger’’ signals at sites of infection and to directly infect APC. Features of viruses that can diminish their potency as vaccine vectors include the presence of virally encoded inhibitors of immunity. These include molecules that block processing and presentation in the MHC class I pathway (such as TAP inhibitors and inhibitors of MHC class I traffic out of the endoplasmic reticulum) and cytokine decoys, to mention a few (260). Deleting immunologic inhibitory genes from recombinant viruses may further enhance their vaccine potency while attenuating their virulence. A major barrier to virus-based vaccination is neutralizing antibodies in preexposed or prevaccinated individuals that inhibit the initial round of infection and replication, thereby quenching their ability to immunize. Individuals who have never been previously exposed to the vaccinating virus generate neutralizing antibody after the first vaccination, thereby precluding subsequent vaccination with the same vector. This finding has led to the concept of cycling different viral vectors in ‘‘prime-boost’’ formats. Dramatic enhancement of immunization potency has been observed in prime-boost formats between both different viruses such as vaccinia followed by fowlpox between DNA vaccines and recombinant viral vaccines (261,262). Among the large number of clinical trials with viral vaccine vectors, the most extensive have involved poxvirus vectors. On the basis of enthusiasm from preclinical experiments, a number of prime-boost studies have been performed using vaccinia followed by fowlpox (263). A phase 3 study in patients with inoperable pancreatic cancer was performed using a vaccinia-fowlpox prime-boost schedule versus chemotherapy or supportive care. The viral vectors incorporated two antigens—carcinoembryonic antigen and MUC-1 and also included ICAM-1, LFA-3, and B7.1 genes to putatively enhance the costimulatory activity of infected DCs (though this has never been proven). The trial was negative. Phase 2 trials with a similar prime-boost regimen for advanced prostate cancer using PSA as the antigen have provided interesting results, but clinical benefit has not been definitively demonstrated. Regulatory hurdles and the inability to vaccinate repetitively are likely to preclude further development efforts for viral vaccines in cancer.

Engineered Bacteria Genetic engineering of intracellular bacteria such as BCG, Salmonella, Shigella, and Listeria has produced a number of interesting and promising vaccines (264–271). In principle

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bacteria that enter APCs may represent a good vehicle for delivery of recombinant antigens. In certain cases, such as Listeria, the bacteria exhibit complex life cycles that involve both phagolysosomal and cytoplasmic stages. Thus, recombinant Listeria monocytogenese engineered to secrete antigens will load the MHC class II processing pathway during the phagolysosomal phase and the MHC class I pathway during the cytosolic phase of the life cycle. In addition, a number of recombinant bacteria actively induce infected APC to secrete proinflammatory cytokines such as IL-12. More recently, recombinant bacteria have been utilized as vectors for delivery of DNA vaccines (269,270). Thus, bacterial vaccines containing plasmids with eukaryotic promoter and enhancer elements driving the antigen gene, result in potent immunization. These results indicate that the bacteria can directly transfer plasmids into eukaryotic transcriptional compartments within infected APC. The Listeria monocytogenes (LM) vectors are among the most promising bacterial vectors being developed for therapeutic vaccination of cancer. Dubensky and colleagues have identified a number of approaches to dramatically attenuate the virulence of LM without diminishing its immunogenicity. One approach is to knock out the ActA and internalin (Inl)B genes of LM (271). Knockout of ActA does not prevent the initial infection of cells with LM but eliminates the capacity for cell-to-cell spread necessary for propagation of LM infections. Knockout of the InlB gene eliminates the capacity of LM to infect hepatacytes while not affecting the capacity of LM to infect APCs. Thus, infection with InlB mutant LM generates strong intrahepatic inflammatory responses with minimal destruction of hepatocytes. ActA/InlB double mutant LM are equivalently immunogenic to wild-type LM, but are 4 to 5 logs attenuated in their virulence. Another approach to virulence attenuation of LM (that is applicable to other bacterial vectors as well) involves the knockout of DNA repair genes together with limited DNA cross-linking using psoralen derivatives. Because the DNA repair system has been knocked out, bacteria can be inhibited from replicating themselves with as few as a single DNA cross-link per bacterial genome. This approach maintains metabolic activity while formally ‘‘killing’’ the bacteria. These killed but metabolically active (KBMA) organisms maintain significant immunogenicity, but have highly attenuated virulence (272).

COMPARISON OF TWO PROSTATE CANCER VACCINES: ONE THAT FAILED IN PHASE 3 AND ONE THAT APPEARS TO HAVE SUCCEEDED IN PHASE 3 While prophylactic vaccine development follows a welldefined and established clinical path, clinical development strategies for cancer vaccines are complex and diverse and must be considered in the context of the vast array of cancer therapeutics under evaluation. Thus, the ultimate failure or success of a cancer vaccine to achieve FDA approval depends as much on clinical trial design and development strategy as on the potency of the vaccine. The reader is referred to a recent review of the regulatory and clinical design issues associated with cancer vaccine development in the context of a number of recently failed phase 3 cancer vaccine trials (273). An illustrative example of the elements of late stage clinical trial development (i.e., phase 3) that impact on success or failure of cancer vaccines comes from the comparison of the Dendreon (Provenge) versus the Cell Genesys prostate cancer vaccine

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(GVAX). The prostate GVAX vaccine phase 3 trial, termed VITAL-1, was terminated after an interim analysis revealed a <30% probability of meeting stated end point of statistical superiority to Taxotere þ prednisone (standard therapy in the United States for advanced hormone resistance prostate cancer) (274). Dendreon has recently presented results of a phase 3 trial, termed Impact, that successfully demonstrated superiority in overall survival of their Provenge vaccine compared with placebo and is applying to the FDA for marketing approval based on these results (275). While extremely different in formulation, both vaccines depend on the loading of DCs with prostate cancer antigens as well as the expansion of DCs with GM-CSF. In the case of GVAX vaccines, two allogeneic prostate cancer cell lines are transduced with the GM-CSF gene and irradiated prior to vaccination. This results in the release of large quantities of paracrine GM-CSF release at the vaccination site, resulting in a huge DC infiltration. As the prostate cancer cells ultimately die subsequent to the irradiation, their antigens are released and taken up by DCs which traffic to the draining lymph nodes and present to T cells. This vaccine is generic (i.e., no patient-specific processing) and contains large numbers of ‘‘shared’’ prostate cancer antigens since the antigen source is two prostate cancer lines. The Provenge vaccine is a patient-specific vaccine that is produced via a rapid and relatively crude purification of DC precursors from the blood of each patient followed by in vitro incubation with a prostatic acid phosphatase (PAP)—GM-CSF fusion protein. The chimeric GM-CSF-PAP antigen serves a dual purpose of targeting the PAP antigen to DC endosomal compartments via binding to their GM-CSF receptor as well as inducing proliferation of the DCs also via the GM-CSF component. Each of these vaccines therefore relies on DC presentation of antigen. However, while GVAX is multivalent, the Provenge vaccine relies on large quantities of a single antigen. Both vaccines were tested in a set of randomized phase 3 clinical trials in men with advanced hormone-refractory prostate cancer. There is no good evidence to favor enhanced potency of either GVAX or Provenge relative to each other since neither has been demonstrated to enhance T-cell responses to prostatespecific or prostate cancer-specific antigens (although GVAX has been shown to induce antibody responses against multiple antigens expressed by the prostate cancer cell lines used for the GVAX). However, significant differences in the clinical trial design resulted in failure of the GVAX vaccine and in contrast, apparent success of the Provenge vaccine. The GVAX vaccine phase 3 trial compared GVAX alone with the standard of care in the United States for men with advanced hormone-refractory prostate cancer—Taxotere þ prednisone (T þ P). T þ P is known to provide a small but real (* 4 months) enhancement in overall patient survival relative to no treatment. For the VITAL-1 trial, Cell Genesys set statistically significant survival benefit relative to T þ P. This relatively high bar was set on the basis of comparisons of survival to historical data with T þ P in an extremely small phase 2 GVAX trial. An interim analysis of VITAL-1 was performed, showing no statistical difference in median survival between the GVAX arm (20.7 months) and the T þ P arm (21.7 months). The treatment related toxicity in the GVAX arm was much lower than the T þ P arm (9% vs. 43% grade 3 toxicity, respectively). An interesting trend was observed in which there was a slightly lower initial death rate in the T þ P arm; however, the curves crossed at about 90 weeks such that the GVAX arm demonstrated superiority to T þ P for patients surviving beyond 90 weeks. A retrospective subset

analysis demonstrated that patients with better predicted survival at the initiation of therapy (based on a set of prognostic parameters termed the Halabi nomogram) demonstrated an even greater advantage to the GVAX arm relative to the T þ P arm once the curves crossed. Because the trial was terminated prematurely, there were not enough patients followed long enough to draw any statistical significance from these trends. The trial was terminated at the interim analysis point precluding further patient follow-up. Dendreon followed a very different strategy of development for Provenge. An initial Provenge phase 3 vaccine trial set as its end point superiority in progression-free survival (as opposed to overall survival) relative to a control arm that involved administration of the patient cells without the PSAGM-CSF chimeric antigen. This control arm therefore did not include T þ P, a therapy with known survival benefit. The trial demonstrated no difference in progression-free survival between the two arms, which would have signified a failed trial given the set end point. However, Dendrion followed the patients and found that the vaccine arm that included the antigen, demonstrated a statistically improved overall survival. On the basis of these results, Dendrion applied for FDA approval on the basis of this revised end point. However, the statistical analysis based on an altered end point was deemed inadequate and marketing approval was denied by the FDA. A subset analysis suggested that patients with earlier stage disease (Gleason score 7) fared better on the vaccine arm than more advanced patients. On the basis of these findings, Dendrion performed another phase 3 trial (IMPACT) with overall survival as an end point. While lower Gleason score was not an absolute inclusion criterion, the overall cohort was skewed toward earlier stage disease (*75% Gleason score 7) as compared with the Cell Genesys VITAL1 patient cohort (*50% Gleason score 7). Further analysis of the characteristics of the enrolled patients and survival curves supports the notion that, within the broad scope of ‘‘advanced hormone resistant prostate cancer,’’ the IMPACT cohort had relatively earlier stage disease at the time of enrollment than the VITAL-1 cohort. They have recently reported a statistically significant mean overall survival benefit of 4.1 months for the vaccine arm (25.8 months) versus the placebo arm (21.7 months). If Dendreon’s BLA is successful, it will represent the first FDA approved therapeutic vaccine for cancer. In summary, comparison of the two development strategies reveals that the difference between success and failure were likely not related to therapeutic efficacy of the vaccines (neither are particularly efficacious as single agents in advanced prostate cancer) but rather two key trial design elements: Dendreon sought to demonstrate superiority of vaccine over placebo and selected patients with relatively less advanced disease while Cell Genesys sought to demonstrate superiority of vaccine over a therapy with known survival benefit (though a much greater toxicity profile). Despite the formal failure of VITAL-1 to meet its stated end points, much information could have been learned and a number of questions could have been answered had the trial not been prematurely terminated: (i) Would the crossover between the GVAX and Taxotere arms have held up as more patients were followed for longer periods? (ii) Did patients receiving Taxotere after receiving GVAX demonstrate a different response rate than those who did not receive prior GVAX vaccination? (iii) Were there any immune correlates of enhanced survival as suggested by the small phase 2 trial

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(such as vaccine induction of humoral responses to certain antigens). Likewise, many questions remain about Provenge: (i) was there any evidence of activation of PAP-specific T cells by vaccination and did this correlate with clinical outcome? (ii) was some of the effect due to adoptive transfer of PAPspecific T cells activated during the in vitro culture? (iii) how much of the enhanced effect relative to ‘‘placebo’’ came from the GM-CSF independent of the PAP antigen? Answers to these and other important questions are critical to advancements in the field.

ENHANCEMENT OF CANCER VACCINE EFFICACY VIA BLOCKADE OF IMMUNOLOGIC CHECKPOINTS AND COSTIMULATORY AGONISTS While the comparative analysis of the Provenge and Prostate GVAX development is highly informative for the ultimate corporate milestone of FDA approval for marketing, it begs the larger question of how to move from small incremental advances in overall survival (i.e., 4 months added to *2 years) to quantum jumps in impact. For cancer immunotherapy, scientific advances described earlier bolstered by preclinical tumor modeling over the past decade have identified a clear path, whose translation into the clinic will require fundamental changes in the culture of cancer therapeutics development. This path involves the coordinate application of vaccines with either antagonists of coinhibitory pathways (often termed immune checkpoints) or agonists of costimulatory pathways as well as agents that alter the immune microenvironment of the tumor itself (Fig. 4). The appreciation of multiple regulatory mechanisms that downmodulate immune responses as well as costimulatory receptors that amplify them provides direct opportunities for therapeutic intervention. For T cells, it is now becoming clear that the TCR signal initiated by antigen recognition is either amplified or muted in a rheostat fashion by multiple costimulatory and coinhibitory signals, respectively. The immunologic role for the coinhibitory receptors is twofold. The first is to inhibit excessive T-cell responses to antigens, thereby limiting tissue destruction once an infection has been eliminated. The

Figure 4 Combinatorial approaches to immunotherapy. Preclinical and preliminary clinical experience suggests that the most effective immunotherapy will combine vaccination with agents that inhibit immune checkpoints that downmodulate the amplitude of T-cell responses (such as CTLA-4), maintain T-cell tolerance (such as regulatory T cells), and inhibit effector immune responses in the tumor microenvironment (such as B7-H1 and B7-H4). Many of these inhibitory signals can be blocked with antibodies against the inhibitory receptor. Abbreviation: CTLA-4, cytotoxic T lymphocyte antigen-4.

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second role is to help maintain tolerance to self-antigens. Tumors help to protect themselves from antitumor immune responses by usurping a number of these inhibitory mechanisms. Because these signals are typically initiated by interaction of cell membrane–bound ligands with receptors on T cells, they are particularly amenable to inhibition with antagonist (blocking) antibodies. Much recent attention has been paid to enhancement of immune responses via antibody blockade of two coinhibitory receptors, CTLA-4 and PD-1. CTLA-4 is upregulated on T cells upon activation and binds B7-1 and B7-2 with higher affinity than the costimulatory receptor—CD28 (276). Thus, CTLA-4 represents a classic example of feedback inhibition of T-cell responses. PD-1 is also upregulated upon T-cell activation (277) and downmodulates T-cell responses when it interacts with its major ligand, B7-H1 (also termed PD-L1) (278) or its minor ligand, B7-DC (PD-L2) (279). Despite their similar structures, analysis of the phenotypes of knockout mice for CTLA-4 and PD-1 demonstrate dramatic differences in their physiology, differences that appear to have important relevance to predicted consequences of antibody blockade of these molecules. CTLA-4 appears to represent a virtual on/off switch for the immune response since CTLA-4 knockout mice spontaneously develop massive immune infiltrates in multiple organs that are lethal by three weeks of age (280). The phenotype of CTLA-4 knockout mice suggests that CTLA-4 blockade with antibodies might dramatically enhance T-cell responses, but with a heavy price of increased autoimmune or hyperimmune responses. Murine studies demonstrated that treatment with a single dose of anti-CTLA-4 antibody resulted in dramatic antitumor effects for immunogenic tumors (i.e., tumors that induced natural immune responses during their growth, but that were insufficient to fully reject them) (281). However, anti-CTLA-4 antibodies as a single agent had virtually no effect on nonimmunogenic tumors. Combination studies between anti-CTLA-4 antibodies and vaccines such as GVAX demonstrated dramatic synergy in the setting of poor immunogenic tumors, suggesting that a vaccine could activate weak T-cell responses against tumor antigens, but that these could be amplified by anti-CTLA-4 blockade (282). Just as predicted by the murine preclinical studies, clinical trials using anti-CTLA-4 as a single agent have demonstrated significant activity in patients with melanoma and renal cell cancer (the two human cancers that appear to have intrinsic immunogenicity) (283). However, immune related toxicities are quite significant, involving dramatic skin rashes and colitis as the predominant side effect, but with less frequent cases of pneumonitis, hepatitis and pan-hypophysitis. Ten years after their introduction into the clinic, the FDA has not yet approved anti-CTLA-4 antibodies as single agents for therapy of any cancer. On the basis of the significant cultural barriers associated with development of two experimental agents, particularly when they are each owned by separate companies (as in the case of GVAX and anti-CTLA-4), no combination vaccine/anti-CTLA-4 trial has been completed and published despite the compelling findings of synergy in preclinical models (first published in 1999). Blockade of the B7-H1-PD-1 axis may be a more promising approach to amplify antitumor immune responses. In particular, upregulation of B7-H1 expression on both tumor cells themselves as well as on nontransformed cells in the tumor microenvironment appears to be a common specific mechanism of immune evasion for many different cancer types (284). For a number of cancer types, levels of B7-H1 expression in the tumor correlate extremely well with prognosis (285), suggesting that

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B7-H1 expression in human cancers plays a true role in tumor biology. Likewise, tumor-specific lymphocytes infiltrating melanomas display high levels of PD-1 on their cell surface. In marked contrast to CTLA-4, the phenotype of both B7-H1 knockout mice and PD-1 knockout mice is mild (286); nonetheless, immunizations, particularly in the context of weak or tolerized immune responses are highly enhanced in PD-1 knockout and B7-H1 knockout mice or in mice treated with blocking anti-PD-1 or anti-B7-H1 antibodies (287,288). Currently, phase 1 trials with anti-PD1 antibodies as well as anti-B7-H1 antibodies are underway with encouraging preliminary evidence for activity in melanoma and renal cancer and importantly, significantly less immune toxicity than observed with anti-CTLA-4 therapy. Preclinical data on antitumor responses suggests that anti-PD-1 and anti-B7-H1 antibodies are active as single agents against immunogenic tumors but show little activity against nonimmunogenic tumors, similar to anti-CTLA-4. Significant synergy between vaccines, including GVAX, and B7-H1/PD-1 blocking antibodies has been reported for nonimmunogenic tumors (289). Additional coinhibitory ligands, such as one termed B7H4 (290,291), have been shown to be upregulated on tumor cells and tumor-associated macrophages as well. Thus, there may be a panoply of inhibitory molecules that can be targeted to enhance antitumor immunity generated by vaccines. Treg represent an additional mechanism for T-cell inhibition. As described earlier, Treg have been shown to highly infiltrate a number of tumor types, both in mouse and human cancer systems. As mentioned earlier, elimination of Treg with antiCD25 antibodies have been shown to enhance vaccine-induced antitumor immunity (104). While no cell surface molecule specific to Treg has yet been identified, some of these molecules include coinhibitory receptors seen on effector T cells such as CTLA-4 and PD-1. On the basis of the recent report by Sakaguchi and colleagues that CTLA-4 is necessary to promote regulatory T-cell function, the concept is emerging that certain molecules may be expressed by both Treg and T effector cells, acting as ‘‘costimulators’’ of Treg-dependent inhibition and cell-intrinsic ‘‘coinhibitors’’ when expressed by effector T cells. Wherry and colleagues, for example, demonstrated that exhausted T cells in the context of chronic viral infection express high levels of PD-1 and LAG-3 (another cell membrane molecule with dual function in promoting Treg inhibition and coinhibiting effector T cells) (98) and demonstrated that blockade of these new molecules can reverse T-cell exhaustion when used in conjunction with each other (292).

SUMMARY The failure of cancer vaccines to generate defined clinical efficacy in randomized phase 3 trials has brought to the fore the question of whether T-cell responses potent enough to be clinically meaningful can be generated. While not feasible for general use, adoptive T-cell transfer studies with tumor-reactive T cells grown ex vivo suggest that antitumor efficacy is indeed achievable if enough T cells with the appropriate effecter activity can be generated. Achieving this in vivo with vaccination remains a challenge because tumor-specific T cells from cancer-bearing patients exist in a hypoactive state and tumors possess multiple resistance mechanisms. Most clinical cancer vaccine trials have been limited by a number of deficiencies, including vaccine formulations that do not effectively activate DCs, poor choice of antigen (for antigen-specific vaccines), poor

choice of patient population (advanced metastatic cancer), inability to repetitively vaccinate (viral vaccines that generate neutralizing humoral immunity after one administration), and failure to combine vaccination with other agents that could enhance vaccine potency. On the clinical side, the major failures of cancer vaccines have come from their application as single agents in advanced disease when preclinical models demonstrate their inability to break established tolerance in that very setting. Ultimate establishment of vaccination as a useful cancer therapy requires that these limitations be addressed. Most importantly, combinatorial approaches that enhance the efficacy of vaccines using agents that enhance T-cell activation status via costimulatory agonists and checkpoint antagonists will maximize the therapeutic potential of cancer vaccines as well as their application in earlier stages of disease, possibly even in preneoplastic states.

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247. Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 1995; 269(5230):1585–1588. 248. Basu S, Binder RJ, Ramalingam T, et al. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 2001; 14(3):303–313. 249. Asea A, Kraeft SK, Kurt-Jones EA, et al. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000; 6(4):435–442. 250. Kuppner MC, Gastpar R, Gelwer S, et al. The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors. Eur J Immunol 2001; 31(5): 1602–1609. 251. Einstein MH, Kadish AS, Burk RD, et al. Heat shock fusion protein-based immunotherapy for treatment of cervical intraepithelial neoplasia III. Gynecol Oncol 2007; 106(3):453–460. 252. Aalamian M, Fuchs E, Gupta R, et al. Autologous renal cell cancer vaccines using heat shock protein-peptide complexes. Urol Oncol 2006; 24(5):425–433. 253. Smith GL, Murphy BR, Moss B, et al. Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza virus infection in hamsters. Proc Natl Acad Sci U S A 1983; 80(23):7155–7159. 254. Panicali D, Davis SW, Weinberg RL, et al. Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin. Proc Natl Acad Sci U S A 1983; 80(17): 5364–5368. 255. Moss B. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci U S A 1996; 93(21):11341–11348. 256. Carroll MW, Overwijk WW, Chamberlain RS, et al. Highly attenuated modified vaccinia virus Ankara (MVA) as an effective recombinant vector: a murine tumor model. Vaccine 1997; 15(4): 387–394. 257. Paoletti E, Taylor J, Meignier B, et al. Highly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC. Dev Biol Stand 1995; 84:159–163. 258. Velders MP, McElhiney S, Cassetti MC, et al. Eradication of established tumors by vaccination with Venezuelan equine encephalitis virus replicon particles delivering human papillomavirus 16 E7 RNA. Cancer Res 2001; 61(21):7861–7867. 259. Elzey BD, Siemens DR, Ratliff TL, et al. Immunization with type 5 adenovirus recombinant for a tumor antigen in combination with recombinant canarypox virus (ALVAC) cytokine gene delivery induces destruction of established prostate tumors. Int J Cancer 2001; 94(6):842–849. 260. Gewurz BE, Gaudet R, Tortorella D, et al. Virus subversion of immunity: a structural perspective. Curr Opin Immunol 2001; 13(4):442–450. 261. Irvine KR, Chamberlain RS, Shulman EP, et al. Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors. J Natl Cancer Inst 1997; 89(21):1595–1601. 262. Ramshaw IA, Ramsay AJ. The prime-boost strategy: exciting prospects for improved vaccination. Immunol Today 2000; 21(4): 163–165. 263. Garnett CT, Greiner JW, Tsang KY, et al. TRICOM vector based cancer vaccines. Curr Pharm Des 2006; 12(3):351–361. 264. Pan ZK, Ikonomidis G, Lazenby A, et al. A recombinant Listeria monocytogenes vaccine expressing a model tumour antigen protects mice against lethal tumour cell challenge and causes regression of established tumours. Nat Med 1995; 1(5):471–477. 265. Thole JE, van Dalen PJ, Havenith CE, et al. Live bacterial delivery systems for development of mucosal vaccines. Curr Opin Mol Ther 2000; 2(1):94–99.

Chapter 85: Cancer-Specific Vaccines 266. Killeen K, Spriggs D, Mekalanos J, et al. Bacterial mucosal vaccines: vibrio cholerae as a live attenuated vaccine/vector paradigm. Curr Top Microbiol Immunol 1999; 236:237–254. 267. Ohara N, Yamada T. Recombinant BCG vaccines. Vaccine 2001; 19(30):4089–4098. 268. Shata MT, Stevceva L, Agwale S, et al. Recent advances with recombinant bacterial vaccine vectors. Mol Med Today 2000; 6(2): 66–71. 269. Sizemore DR, Branstrom AA, Sadoff JC, et al. Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 1995; 270(5234):299–302. 270. Darji A, Guzman CA, Gerstel B, et al. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 1997; 91(6): 765–775. 271. Brockstedt DG, Giedlin MA, Leong ML, et al. Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proc Natl Acad Sci U S A 2004; 101(38):13832–13837. 272. Brockstedt DG, Bahjat KS, Giedlin MA, et al. Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity. Nat Med 2005; 11(8):853–860. 273. Finke LH, Wentworth K, Blumenstein B, et al. Lessons from randomized phase III studies with active cancer immunotherapies–outcomes from the 2006 meeting of the Cancer Vaccine Consortium (CVC). Vaccine 2007; 25(suppl 2):B97–B109. 274. Abstract no 150, ASCO 2009 Genitourinary Cancers Symposium, Orlando, FL, February 26–28, 2009. 275. Abstract no 1480, Annual Urologic Association Meeting, Chicago, IL, April 24–28, 2009. 276. Chambers CA, Kuhns MS, Egen JG, et al. CTLA-4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol 2001; 19:565–594. 277. Ishida Y, Agata Y, Shibahara K, et al. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death. EMBO J 1992; 11(11):3887–3895. 278. Dong H, Zhu G, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999; 5(12):1365–1369. 279. Tseng SY, Otsuji M, Gorski K, et al. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J Exp Med 2001; 193(7):839–846. 280. Waterhouse P, Penninger JM, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 1995; 270(5238):985–988.

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281. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science 1996; 271(5256): 1734–1736. 282. van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyteassociated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med 1999; 190(3): 355–366. 283. Phan GQ, Yang JC, Sherry RM, et al. Cancer regression and autoimmunity induced by cytotoxic T lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma. Proc Natl Acad Sci U S A 2003; 100(14):8372–8377. 284. Dong H, Strome SE, Salomao DR, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med 2002; 8(8):793–800. 285. Thompson RH, Gillett MD, Cheville JC, et al. Costimulatory B7H1 in renal cell carcinoma patients: indicator of tumor aggressiveness and potential therapeutic target. Proc Natl Acad Sci U S A 2004; 101(49):17174–17179. 286. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999; 11(2): 141–151. 287. Goldberg MV, Maris CH, Hipkiss EL, et al. Role of PD-1 and its ligand, B7-H1, in early fate decisions of CD8 T cells. Blood 2007; 110(1):186–192. 288. Tsushima F, Yao S, Shin T, et al. Interaction between B7-H1 and PD-1 determines initiation and reversal of T-cell anergy. Blood 2007; 110(1):180–185. 289. Li B, VanRoey M, Wang C, et al. Anti-programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor–secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors. Clin Cancer Res 2009; 15(5):1623–1634. 290. Sica GL, Choi IH, Zhu G, et al. B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 2003; 18(6): 849–861. 291. Kryczek I, Zou L, Rodriguez P, et al. B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J Exp Med 2006; 203(4):871–881. 292. Blackburn SD, Shin H, Haining WN, et al. Coregulation of CD8þ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 2009; 10(1):29–37.

86 Vaccines Against Human Papillomaviruses Kathrin U. Jansen SVP Early Phase Programs, Wyeth Vaccine Research, Pearl River, New York, U.S.A.

Ian H. Frazer Diamantina Institute for Cancer Immunology and Metabolic Medicine, The University of Queensland, Woolloongabba, Queensland, Australia

INTRODUCTION Cervical cancer is one of the more common causes of cancer death among women worldwide, and it is initiated by infection with a limited subset of human papillomaviruses (HPV). Prophylactic vaccines, based on recombinant HPV virus-like particles (VLPs), and designed to induce HPV-neutralizing antibody, have recently been introduced into clinical practice following extensive clinical trials. The vaccines provide lasting immunity against infection with the HPV genotypes that they incorporate, and against the premalignant conditions caused by these infections. If widely deployed, these vaccines could reduce the global burden of cervical cancer by over 70%, and the overall cancer burden by over 4%. While these vaccines have been already proven highly successful, there may still be room for development of simpler technologies more adapted for use in the developing world and for prophylactic vaccines with broader coverage against the HPV types currently in circulation. Therapeutic vaccines, based on papillomavirus (PV) nonstructural proteins, are in early-phase clinical trials and several years from the clinic. This chapter reviews current knowledge of HPV vaccines, discusses vaccine deployment, and highlights where the field may progress over the next few years.

THE GLOBAL BURDEN OF HPV-ASSOCIATED DISEASE HPV infections are common, and are responsible for about 5% of the global burden of human cancers and at least 0.5 million cancer deaths annually (1). HPVs are nonlytic double-stranded DNA viruses that replicate in epithelial cells using the programmed reproduction and differentiation of the epithelium to control and complete their own replication cycle (2). They can be divided broadly into groups according to their preferred site of infection (genital or nongenital skin), and their ability to cause hyperkeratotic skin lesions, commonly referred to as warts. Within these broad groups, further subdivision based on genetic sequence analysis defines clades of viruses producing similar pathology (3). HPV clades A7 and A9 are the major causes of HPVassociated cancers of the anogenital tract. Viruses of these clades are mucosotropic, infecting genital and (to a lesser extent) oropharyngeal, squamous epithelia. They are responsible for over 98% of cervical cancer, and between 30% and 50% of other

anogenital squamous cancers (vulval, vaginal, anal, penile) (4). They are also associated with 10% to 20% of oropharyngeal cancers and may contribute to the etiology of esophageal cancer (5). Association of these viruses with squamous cancers at other sites remains a subject of active investigation. Because of the significant cancer burden caused by persistent infections with HPVs of these clades, they are referred to as ‘‘high-risk’’ HPV types, although more than 90% of infections with these viruses resolve spontaneously in immunocompetent subjects over a period of one to two years (6). HPV16 is responsible for about 50% of HPV-associated cancers, and HPV18 about 20%: these viruses seem particularly prone to initiate cancers (7). Other viruses from the high-risk anogenital clades are each isolated from less than 5% of anogenital cancers, yet they make up a larger percentage of the overall burden of genital HPV infections. Simultaneous infection with multiple genital HPV genotypes is found in 5% to 10% of infected patients. HPV clade A10 is also mucosotropic, and viruses from this clade produce hyperkeratotic lesions of the genital tract epithelium (genital warts). Because these lesions generally resolve spontaneously and rarely progress to cancer, viruses from this clade are referred to as ‘‘low-risk’’ HPV types. The overall contribution of these viruses to human disease is nevertheless substantial. Treatment for genital warts is a major consumer of health care resources in many countries (8). Additionally, the rare but serious condition of recurrent respiratory papillomatosis (9) is caused by viruses of this clade, transmitted from mother to infant at the time of birth. HPVs of the nongenital clades are responsible for skin warts and for a percentage of skin cancer, particularly in immunosuppressed individuals. The extent to which skin cancers can be attributed to HPV infection is highly controversial (10). Vaccines for nongenital HPVs are not under development at present, and they will not be further discussed in this chapter, though the potential utility of such vaccines should not be forgotten.

NATURAL IMMUNITY TO PAPILLOMAVIRUS CAPSIDS Productive infection with genital and skin genotypes of PV is generally self-limiting, and resolution of infection, which occurs over months to years, requires an immunocompetent host. Measurable humoral (11) and cellular (12) immune

Chapter 86: Vaccines Against Human Papillomaviruses

responses to the nonlytic HPV infection are generally slow to appear and less robust compared with those seen with lytic or systemic virus infections. Deficits in cell-mediated immunity reduce the clearance of existing infections and increase the risk of progression of infection to malignancy (13,14). The extent to which prior infection protects against subsequent exposure to the same or other genotypes of HPV remains unresolved despite extensive research. However, infection with cutaneous PVs is universal in early childhood, and is rare among adults, including those involved with the care of young children. Similarly, infection of the genital tract is most common among those recently sexually active (15), and is not as common among older sexually active individuals, including sex workers with high-exposure risk (16). Thus, while there are some epidemiological data for HPV16 to suggest that prior infection may convey only partial protection on subsequent exposure, a broad conclusion can be entertained that exposure to PVs eventually protects against further infection in immunocompetent subjects.

HPV VACCINE TECHNOLOGIES PVs are icosahedral viruses comprising 360 copies of the major L1 capsid protein, assembled from 72 pentameric rings (17). HPV vaccines can be broadly divided into those designed to prevent HPV infection and those designed to treat infection. Although PVs were among the first demonstrably infectious filterable agents (18), a major problem with developing a vaccine to prevent PV-associated disease has been production of sufficient virus material to be the basis of a vaccine. PVs cannot easily be grown in tissue culture, and therefore recombinant DNA technology was required to produce PV capsids that are capable of inducing neutralizing antibody and thus prevent PV infection. Existing vaccines to prevent HPV infection are based on such a VLP technology (19,20) where the major capsid protein of the virus is expressed in eukaryotic cells and self assembles to produce empty viral capsids. Other approaches to immunoprophylaxis currently being evaluated include vaccines based on the minor (L2) capsid protein (21), and on assemblies of L1 protein that are less than the entire capsid (22), including pentamers and smaller arrays of pentamers of L1. Therapeutic vaccines under development are based on viral nonstructural proteins like E1, E2, E6, and E7 expressed in infected cells (23), and in premalignant and malignant lesions arising from HPV infection.

PRODUCTION OF PAPILLOMAVIRUS CAPSIDS Recognition that the capsid proteins of PV self-assemble into VLPs, if expressed in a eukaryotic expression system (19,20), has facilitated development of particle-based prophylactic vaccines against HPV infection and disease. The prototype HPV16 L1 clone had a nonconservative mutation (20), not found in subsequent clinical isolates, that prevented assembly of VLPs. Production of VLPs in mammalian cells was inefficient, and production on a larger scale was facilitated by expression of L1 protein in insect cells using recombinant baculovirus (20,24), and in yeast (25,26). These observations paved the way for early vaccine studies using species-specific PVs (27,28). Expression of PV capsid protein in Escherichia coli produces small amounts of VLPs (29), following denaturation and renaturation from amorphous inclusion bodies. Significant amounts of L1 protein assemble into L1 pentamer capsomers (30), which retain at

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least some of the structures necessary to induce virusneutralizing antibody. When L1 is expressed in Salmonella typhimurium, VLPs are also observed (31). However, the practical utility of a particle or capsomer production system based on prokaryotic systems remains uncertain. Stability of VLPs once assembled is critical for the development of a successful vaccine. Current vaccines require storage at 28C to 88C. The requirement for a cold chain for vaccine deployment is not a concern for the developed world but may slow the use of these vaccines in the developing world. Technologies to develop room temperature–stable vaccines are available and could be considered to enhance uptake in the developing world.

EFFICACY DATA Trials to demonstrate the safety and efficacy of vaccines designed to prevent infection with genital HPV infections, and also to prevent the premalignant consequences of these infections, have focused on HPV6/11/16/18. These studies have largely been undertaken in young sexually active women, though more recently, studies have been extended to include women up to the age of 45. Phase I studies demonstrated safety and immunogenicity in human subjects, and phase II studies extended these data to include data on prevention of infection. Pivotal licensing studies addressed efficacy in prevention of clinically relevant HPV-associated disease, including cervical and other anogenital precancer [cervical intraepithelial neoplasia 2 and 3 (CIN2/3)] and genital warts. Phase I studies, in subjects with and without HPV infection, confirmed the safety and immunogenicity of VLP-based vaccines, given intramuscularly with or without adjuvant (32–35). Three administrations of VLPs with adjuvant-induced peak antibody levels at least 10 times higher than those seen in subjects naturally infected with HPV, and levels of antibody above those produced by natural infection were sustained for at least five years post vaccination. In human clinical trials, a dose of 20 to 40 mg, given on three occasions, gave optimal antibody titers. Phase II studies (36–38) addressed immunogenicity and ability to prevent infection in women naı¨ve at recruitment to the HPV types in the administered vaccine, and generally demonstrated near 100% efficacy in prevention of acquisition of new high-risk HPV infections among sexually active young women with no markers (HPV antibody or DNA) of past or current infection with a high-risk HPV type. Phase III studies (39–44) (Table 1) have further demonstrated near 100% efficacy at preventing HPV infection and associated anogenital disease due to vaccine HPV types, in young sexually active women in ‘‘according to protocol’’ analyses, with efficacy data extended to three years in most studies. The efficacy analyses were conservative in that new cytological or clinical abnormalities appearing during the trials that included a vaccine HPV type in addition to a nonvaccine type HPV in the same lesion were counted as vaccine failures, even though the lesion could have been caused by the nonvaccine type alone. For the phase III efficacy studies, young sexually active women were recruited without regard to prior HPV infection status. There was no evidence that immunization impacts the natural history of existing HPV infection, which regressed or progressed in similar rates in vaccine and placebo recipients (46). Therefore, ‘‘intention to treat’’ efficacy of the vaccines for prevention of all HPV-related disease, including the 30% of disease due to nonvaccine types, and including women already infected with a vaccine HPV type

6/11/ 16/18 6/11/ 16/18 6/11/ 16/18

6/11/ 16/18 16/18

16/18

Koutsky (44)

Joura (41)

Harper (42)

Paavonen (40)

7788

481

7811

2241 2261

10291

6087

Vaccine

7838

470

7785

2258 2279

10292

6080

Control

15–25

15–25

16–26

16–24

16–26

15–26

Age range (Yr)

1.25

4–5

3

3

3

3

Follow up (Yr) CIN2/3 AIS CIN2/3 AIS CIN2/3 AIS GW VIN VAIN VIN 2/3 VAIN 2/3 CIN CIN2/3 CIN CIN2/3

End points

–

98 (86–100) 99 (93–100) 100 (94–100) 100 (94–100) 100 (72–100) –

PP (spec HPV)

100(42–100) 100(
8–100) 89(59–99) 90 (53–99)

–

–

–

–

MITT (spec HPV)

–

44 (26–58) 44 (31–55) 55 (40–66) 73 (58–83) 71 (37–88) –

ITT (spec HPV)

Vaccine efficacya

17 (1–31) 18 (7–29) 20 (8–31) 34 (15–49) 49 (18–69)

ITT (any HPV)

PP, per protocol; protection against development of anogenital disease caused by HPV of a vaccine type among subjects without evidence of prior or current infection with that HPV type at the completion of a full immunization schedule per protocol. MITT, modified intention to treat; analysis of efficacy for vaccine type HPV. ITT (spec HPV), Intention to treat; protection observed against anogenital disease caused by HPV of a vaccine type, regardless of subject’s prior or current HPV infection status at recruitment, provided that no HPV-associated disease was present at recruitment. a Mean values with 95% confidence limits are shown. Abbreviations: CIN, cervical intraepithelial neoplasia; AIS, adenocarcinoma in situ; GW, genital warts; VIN, vulval intraepithelial neoplasia; VAIN, vaginal intraepithelial neoplasia. Source: Adapted from Ref. 45.

Garland (39)

Ault (43)

Vaccine

Study

No. of subjects

Table 1 Phase III Efficacy Study Outcomes for the Bivalent and Quadrivalent HPV Vaccines

956 Jansen and Frazer

Chapter 86: Vaccines Against Human Papillomaviruses Table 2 Protection Against Premalignancy Associated with Various HPV Types Among Recipients of the Quadrivalent HPV Vaccine No. of cases of CIN 2/3 or AIS by type HPV type Four vaccine types: HPV6/11/16/ 18 10 Non-vaccine oncogenic types: HPV31/33/35/39/ 45/51/52/56/58/59 Nonvaccine HPV A9 Species HPV31 HPV33 HPV35 HPV52 HPV58 Nonvaccine HPV A7 species HPV39 HPV45 HPV59 HPV51 HPV56

Vaccine (n ¼ 4616)

Placebo (n ¼ 4675)

Efficacy (95%CI)

0

52

100 (93, 100)

38

62

38 (6, 60)

26

48

45 (10, 68)

5 7 2 12 9 8

21 13 1 16 13 15

46 (–35, 80)

2 3 4 8 5

6 2 7 10 10

Abbreviations: CIN, cervical intraepithelial neoplasia; AIS, adenocarcinoma in situ. Source: From D. Brown, Oral presentation, ICAAC 2007.

but without disease at recruitment, has been as low as 17% (Table 1), emphasizing the need for programs of immunization to be undertaken before exposure to virus for the full public health benefit. While the major efficacy studies have been undertaken in younger women, one vaccine efficacy study with the quadrivalent vaccine has shown at least 90% protection against infection and anogenital disease in previously uninfected women aged 25 to 45 years (Luna, J. Oral presentation, 24th International Papillomavirus workshop, Beijing, 2007). Further analysis of the major efficacy studies suggests that immunization with HPV16 and HPV18 VLPs provides some protection against infection with high-risk HPVs of other, nonvaccine HPV types, and against disease attributable to these infections. In a pivotal trial of a bivalent HPV16/18 vaccine, some protection was also seen against HPV45 and HPV33 infection (42). For the quadrivalent HPV6/11/16/18 vaccine (Table 2), HPV naive subjects showed a 27% protection against new anogenital premalignancy (CIN2/3 and AIS) associated with 10 nonvaccine HPV types (Brown, D. Oral presentation, ICAAC 2007). In each of these studies actual case numbers were quite low, making the degree of added protection against cervical malignancy hard to assess. However, it seems unlikely to be sufficient to alter the need to continue assessment of women for cervical precancer through conventional screening where such programs are offered.

ANTIBODIES AS A SURROGATE MARKER FOR PROTECTION Most viral vaccines work by inducing neutralizing antibody to conformational determinants on the surface of the virion. Antibody to conformational determinants on the PV capsid is

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sufficient to convey protection against challenge with live PV when passively transferred in either a dog or a rabbit model (48). Serum immunoglobulin (IgG) antibody seems sufficient to convey protection. Murine and in vitro models of HPV infection (48,49) suggest that HPV enters the epithelium through microabrasions and binds to basement membrane proteoglycans, where it is thought to undergo a conformational change exposing a secondary receptor that facilitates entry into epithelial cells. Neutralizing monoclonal antibodies recognizing conformational determinants on the capsid block infectivity either by blocking virus binding or alternatively by interfering with the conformational change. It is thus likely that the major protection against PV infection may be due to exudation of serum antibody at the site of microabrasions. An immunological surrogate marker for vaccine-induced protection against HPV infection and disease would be useful for bridging studies, and for assessing duration of protection postimmunization. There are no standardized assays for antibody to HPV virions, though the World Health Organization in collaboration with the National Centre for Biological Standards is attempting to produce a standardized serology assay for HPV16 (50,51). Antibodies to HPV are measured in human serum by a range of in vitro and in vivo assays (52–55), including binding of antibody to virus or VLPs, blocking of binding of known neutralizing antibody to VLPs, neutralization of HPV virions mixed with foreskin epithelial cells and transplanted under the renal capsule of a nude mouse, and neutralization of pseudovirions comprising the viral capsid and a reporter gene construct that can be used to report infection of a susceptible cell line. Each assay measures different antibody specificity and function, and a different proportion of the total virus-neutralizing capacity of a serum. Thus antibody titers, even if measured in the same assay, can only be valid for antibody to one HPV type, induced by one vaccine product. Comparative titers, even measured in a standardized assay, are therefore most meaningful as a measure of the relative immunocompetence of different populations immunized with the same vaccine. Antibody assays have thus been used as bridging assays for the introduction of vaccine into younger age groups (male and female children aged 9–15 years) where new infection with HPV is less common and efficacy studies are also not feasible because of the young age. Individuals of the 9- to 15-year-old group produced on average higher levels of antibody than the 16- to 24-year-old women in whom vaccine efficacy studies were undertaken, suggesting that the younger age individuals will also be protected against HPV infection. Antibody assays have also been used to compare immunogenicity of different adjuvants delivered along with the same vaccine. A bivalent HPV16/18 vaccine formulated with aluminum hydroxide and monophosphoryl Lipid A appeared more immunogenic than the same vaccine formulated with aluminum hydroxide alone (38). However, because of the limitations in interpretation of antibody assays outlined above, the clinical significance of any measured differences in vaccine immunogenicity or induced antibody for degree or duration of protection against HPV infection will always be uncertain. Despite these limitations, antibody levels have also been offered as a measure of duration of protection following vaccination. Antibody levels peak after three doses of vaccine at levels at least 20 times higher than those seen after natural infection. While they fall significantly immediately after immunization, they subsequently plateau; and five years after immunization

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antibody titers still remain at levels well above those seen after natural infection. Most importantly, immunized subjects remain protected against infection during that time. Importantly, immunological memory is retained, as reimmunization of immunized young women five years after their primary immunization resulted in a substantial boost to antibody titers following a single immunization not observed in nonimmune subjects (56). However, the longevity of protection ultimately will need to be established through detailed monitoring of immunized individuals for cervical precancer and genital warts. It is noteworthy that for hepatitis B vaccines, protection against disease can persist even in the presence of antibody below a minimum protective level, providing that antibody response was initially induced by the vaccine.

SAFETY DATA Common adverse events after vaccination can be assessed through observations in placebo-controlled efficacy studies, whereas less-frequent events can be effectively monitored only through postmarketing surveillance. Local reactogenicity at the site of immunization, and systemic malaise, has been mild with the VLP-based HPV vaccines throughout the clinical studies, though it has generally been observed with a slightly greater frequency than with the corresponding adjuvant alone. Significant local redness and swelling was seen more frequently in recipients of monophosphoryl Lipid A adjuvanted vaccine than in placebo recipients (40,42). No serious vaccine adverse events possibly attributable to vaccination were seen for either the bivalent or the quadrivalent vaccine. Over 18 million doses of the quadrivalent vaccine have been delivered to young women subsequent to vaccine licensure. A few cases of vaccine-associated urticaria and allergy have been described (57). However, serious adverse events have not been seen with more frequency in vaccine recipients than would be expected in an unvaccinated age-matched group. As with all new vaccine products, ongoing surveillance through vaccine registries and otherwise should be and is being conducted.

VACCINE DEPLOYMENT The quadrivalent HPV vaccine is licensed for use in over 80 countries, and the bivalent HPV vaccine in several countries, with more licensures pending for both products. Optimal vaccine delivery involves three doses of vaccine given over six months, though accelerated vaccine delivery schedules over four months are being used in some countries. The age range for immunization of young women can differ by the country in which the vaccine is licensed but generally reflects the ages for which safety, immunogenicity, and efficacy data are available. Australia and some European Economic Community countries have chosen to introduce mass immunization campaigns of preteen girls on an ongoing basis at government expense. These have been given along with short-term ‘‘catch-up’’ immunization programs of girls up to the age of 18 to 25 years, as modeling suggests that vaccination programs will have the most immediate impact on prevention of cervical precancer detected through existing screening programs if such catch-up immunization programs are undertaken (58). Wherever screening programs for cervical cancer exist, they will be continued in the immunized population, in part as surveillance for disease caused by nonvaccine HPV types, and in part as monitoring of continued efficacy of the vaccine program itself.

As discussed above, efficacy has now also been demonstrated for the quadrivalent vaccine in older women where there is an ongoing incidence of new high-risk HPV infections, although with reduced frequency with increasing age. Currently, most cervical disease in older women results from persistence of already existing infection. This underscores the importance for older women, whether vaccinated or not, to continue cervical cancer screening. It is likely that immunization of older women will be elective, and will reflect the preferences of the women, and the incidence of new HPV infection by age in the community. Some countries have also licensed the vaccine for use in young men, on the basis of safety and immunogenicity data alone, even though ongoing efficacy studies in men have not concluded yet. The greatest public health benefit from immunization with HPV vaccines will come if these vaccines are given to young men and women before sexual debut, as the vaccines are prophylactic but not therapeutic (46). It has been demonstrated in the past that gender-specific vaccination as exemplified by early Rubella vaccination campaigns restricted to girls only were not as effective (59), leading subsequently to vaccination recommendations for both boys and girls. It is also widely accepted that vaccines work well because they can induce herd immunity. Efficacy studies in men, presuming that they show at least some benefit, should encourage the use of these vaccines globally for both males and females to maintain herd immunity, protect sexual partners, and reduce the incidence of HPV-associated cancer in both genders.

BARRIERS TO VACCINE USE Two years after licensure of the quadrivalent HPV vaccine, the vaccine appears to be successfully introduced in many developed countries. Nevertheless, significant barriers remain to even broader distribution. One barrier is the need for ongoing education of decision makers, health care providers, and potential vaccine recipients about vaccine benefits and use. In many industrialized and transitional countries, routine childhood and infant vaccinations are administered by general practitioners and pediatricians, who have little direct experience of women’s health issues, including cervical cancer. HPV vaccines are administered to young adolescents and young women, and pediatricians or general practitioners confronted with an adolescent and their parent may not be comfortable in discussing the vaccine’s benefits, or the potential vaccinee’s sexual behavior, and may therefore not promote the vaccine. Should HPV vaccines be also licensed for use in boys and adolescent males, this issue may become even more important. A second and related barrier relates to concerns about vaccine safety. With the increased use of the Internet to disseminate information, whether accurate or not, many decision makers are left with doubts whether the vaccines are actually safe. The anti-vaccine lobby adds to these doubts with unsubstantiated and subjective ‘‘information.’’ To overcome this barrier, companies, regulatory agencies, and state health boards may need to be more vocal in disseminating factual and objective information about vaccine safety in venues that are easily accessible and distributed to those that make decisions about vaccination. A third and formidable barrier is the high cost of the vaccine, which impacts the programmatic introduction of HPV

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vaccines in developing countries. This issue is not unique to HPV vaccines. Over 80% of the cervical cancer burden is in developing countries that do not have routine cervical cancer screening programs and have health care budgets insufficient to enable routine HPV vaccination. To introduce HPV vaccines to these countries requires public-private partnerships, political will, and clever strategies that will present win/win situations to the countries, the vaccine manufacturers, and the funding agencies. One could envision strategies that allow a country to license ultimately the vaccine technologies for future production and commercialization in select markets. Until there is developing country vaccine production (or if attempts to achieve production prove too challenging), subsidized vaccine supplies could be made available. An alternative strategy would be to provide subsidized bulk vaccine supplies to appropriate vaccine manufacturers in developing countries to take care of formulation, fill, release, regulatory approval, and distribution across developing world countries. A further barrier, particularly in the United States, is that vaccine providers (pediatricians, family practitioners, internists) have to buy the HPV vaccine to make it available for their patients. This presents problems with vaccine storage and with cash flow, as providers pay for vaccine on supply, and are only reimbursed by insurers after the vaccine is administered.

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more expensive. Addition of an additional six HPV types would increase cancer coverage to more than 90%, presuming that there was no interference between the different HPV types in the vaccine. Alternate strategies to broaden vaccine coverage by including the L2 proteins has shown some preclinical promise but needs to be substantiated clinically. Validation of different delivery schedules will likely be undertaken, including childhood immunization with adolescent boosting (which may prove more practical for the developing world), or a two-dose immunization regimen that might give sufficient protection against disease in immunocompetent subjects. For the moment, however, the major challenge remains to develop and validate effective strategies for deployment of the currently available vaccines, particularly in the developing world.

ACKNOWLEDGMENTS We like to acknowledge the many individuals in the HPV field that have contributed scientifically in one way or another to an understanding of HPV disease, epidemiology, and protective immunity, as well as to the development of successful prophylactic vaccines.

DECLARATION OF CONFLICT OF INTEREST HPV THERAPEUTIC VACCINES Development of immunotherapy to alter the course of existing HPV infections has not progressed with the same speed as occurred with the development of the prophylactic HPV vaccines. Partly, this represents a lack of knowledge about how best to produce effective immunotherapy for any disease. It is likely that cytotoxic T cells directed against viral antigens will assist in clearing persisting HPV infection, as they do in the process of natural clearance of the majority of viral infections. However, it remains unclear why a small percentage of highrisk HPV infections become persistent, and whether this represents host inability to mount a relevant response or viral evasion of host immunity. There are also no effective immunotherapeutic vaccines to eradicate persisting infections in humans. Repeated exposure to antigen switches induced immunity from T helper 1 (Th1) type, favoring cytolytic responses to Th2 type and may pose a challenge for effective immunotherapy in persistently infected patients (60). Despite these theoretical and practical problems, efforts persist to develop HPV-specific immunotherapy, perhaps because the viral antigens are well known and the target lesions are well understood, facilitating clinical trials. Several clinical trials have been undertaken, and have been reviewed elsewhere (61,62). However, heretofore, none have shown substantial benefit for patients with persistent HPV infection or with cervical cancer.

FUTURE PROPHYLACTIC VACCINE DEVELOPMENTS Vaccines based on VLP technology have proven remarkably successful at preventing cervical disease associated with the HPV types present in the vaccine if vaccine is delivered to women before acquisition of infection. Broadening the coverage of HPV types to include those responsible for much of the burden of anogenital cancer not attributable to HPV16 or HPV18 infection would likely increase vaccine utility, though not necessarily the cost benefit if the vaccine were proportionately

IHF, as named inventor on patents relating to HPV VLPs, derives royalty income from the sale of HPV vaccines mentioned in this article. IHF consults for Merck, GlaxoSmithKline, and CSL Ltd. KUJ—as named inventor on patents relating to the development of Merck’s prophylactic HPV vaccine.

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32. Evans T G, Bonnez W, Rose RC, et al. A phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers. J Infect Dis 2001; 183: 1485–1493. 33. Harro CD, Pang YY, Roden RB, et al. Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 viruslike particle vaccine. J Natl Cancer Inst 2001; 93:284–292. 34. Zhang LF, Zhou J, Chen S, et al. HPV6b virus like particles are potent immunogens without adjuvant in man. Vaccine 2000; 18: 1051–1058. 35. Fife KH, Wheeler CM, Koustsky LA, et al. Dose-ranging studies of the safety and immunogenicity of human papillomavirus type 11 and type 11 and type 16 virus-like particle vaccine in young healthy women. Vaccine 2004; 22:2943–2952. 36. Koutsky LA, Ault KA, Wheeler CM, et al. A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 2002; 347:1645–1651. 37. Villa LL, Ault KA, Giuliano AR, et al. Immunologic responses following administration of a vaccine targeting human papillomavirus Types 6, 11, 16, and 18. Vaccine 2006; 24(27–28): 5571–5583. 38. Giannini SL, Hanon E, Moris P, et al. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine 2006; 24:5937–5949. 39. Garland SM, Hernandez-Avila M, Wheeler CM, et al. Quadrivalent vaccine against human papillomavirus to prevent anogenital diseases. N Engl J Med 2007; 356:1928–1943. 40. Paavonen J, Jenkins D, Bosch FX, et al. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007; 369:2161–2170. 41. Joura EA, Leodolter S, Hernandez-Avila M, et al. Efficacy of a quadrivalent prophylactic human papillomavirus (types 6, 11, 16, and 18) L1 virus-like-particle vaccine against high-grade vulval and vaginal lesions: a combined analysis of three randomised clinical trials. Lancet 2007; 369:1693–1702. 42. Harper D M, Franco EL, Wheeler CM, et al. Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial. Lancet 2006; 367:1247–1255. 43. Ault KA. Effect of prophylactic human papillomavirus L1 virus-like-particle vaccine on risk of cervical intraepithelial neoplasia grade 2, grade 3, and adenocarcinoma in situ: a combined analysis of four randomised clinical trials. Lancet 2007; 369:1861–1868. 44. The Future 2 Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007; 356:1915–1927. 45. Kahn JA, Burk RD. Papillomavirus vaccines in perspective. Lancet 2007; 369:2135–2137. 46. Hildesheim A, Herrero R, Wacholder S, et al. Effect of human papillomavirus 16/18 L1 viruslike particle vaccine among young women with preexisting infection: a randomized trial. JAMA 2007; 298:743–753. 47. Ghim S, Newsome J, Bell J, et al. Spontaneously regressing oral papillomas induce systemic antibodies that neutralize canine oral papillomavirus. Exp Mol Pathol 2000; 68:147–151. 48. Day PM, Thompson CD, Buck CB, et al. Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition. J Virol 2007; 81:8784–8792. 49. Roberts JN, Buck CB, Thompson CD, et al. Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med 2007; 13:857–861. 50. Ferguson M, Heath A, Johnes S, et al. Results of the first WHO international collaborative study on the standardization of the detection of antibodies to human papillomaviruses. Int J Cancer 2005; 118(6):1508–1514. 51. Pagliusi SR, Garland SM. International standard reagents for HPV detection. Dis Markers 2007; 23:283–296.

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87 Vaccines Against Alzheimer’s and Other Neurodegenerative Diseases Roy M. Robins-Browne Department of Microbiology and Immunology, University of Melbourne, Murdoch Childrens Research Institute, Royal Children’s Hospital, Victoria, Australia

Colin L. Masters Centre for Neuroscience, University of Melbourne, The Mental Health Research Institute, Victoria, Australia

INTRODUCTION Alzheimer’s disease (AD) is a progressive neurological disorder that imposes an enormous burden on society (1). Unlike some other progressive neurological diseases, such as subacute sclerosing panencephalitis, progressive multifocal leukoencephalopathy, and Creutzfeldt-Jakob disease (CJD), there is no evidence to suggest that AD is the result of an infection. Hence it is all the more remarkable that AD may be the first of the chronic degenerative neurological diseases to be amenable to specific intervention through therapeutic and preventative immunization. In common with other degenerative diseases of the nervous systems, AD is characterized by gradually evolving progressive neuronal degeneration, which leads to global impairment of memory and other cognitive functions in a way that interferes with normal social or occupational performance (dementia) (1). As the disease progresses, behavioral changes occur including passivity and withdrawal, accompanied by impairment of attention, judgment, recognition, insight, and language. Later, agitation, suspiciousness, wandering, and hallucinations may occur. In the final stages, patients enter a vegetative state and become mute and uncomprehending. The condition was formally described in 1907 by Alois Alzheimer, who reported the clinical and pathological findings of a disease in a 57-year old woman with progressive dementia (2,3). Although senile dementia (dementia occurring in people older than 65 years) was well known at the time, this patient was notable because of her relatively young age at onset, and because an autopsy revealed large numbers of amyloid plaques and neurofibrillary tangles in her brain. This unique combination of clinical and pathological features came to be known as AD, a term which was originally limited to ‘‘presenile’’ dementia, but now is applied to all patients with these clinicopathological features regardless of age. Although AD may affect people in younger age groups, most patients are aged over 65 with the prevalence increasing with advancing age, such that up to 10% of the population over 65, and 50% over 80 may be affected (1). The duration of the disease from the time of diagnosis to death may exceed 20 years, although the average length is from 8 to 10 years. In 1993, Rice et al. (4) estimated the cost of AD in the United States

to be US$63.3 billion, made up from direct financial outlays, such as nursing care, and indirect costs, including loss of productivity of the patients themselves and the family members who care for them. As people born in the population boom which followed World War II reach their 60s and 70s, these costs are poised to escalate considerably unless something is done to reduce the incidence of AD.

PATHOLOGY The hallmark pathological lesions of AD are the extracellular amyloid plaques and intracellular neurofibrillary tangles originally described by Alzheimer. The characteristic plaques are distributed throughout the cerebral cortex at a high density, and are made up mainly of degenerated neuronal processes (neurites) and deposits of b-amyloid (Ab) protein, which also occurs within the walls of cerebral blood vessels. Neurofibrillary tangles are intraneuronal structures that appear as paired helical filaments when viewed by electron microscopy. They are particularly evident in medial temporal lobe structures, including the hippocampus, a region of the brain that is important for memory (5). Neurons that contain these tangles have a tendency to lose their synaptic connections and eventually die leaving ‘‘ghost tangles.’’ Although the accumulation of amyloid plaques and neurofibrillary tangles may occur in the brain of non-demented persons, both types of lesion occur at a far higher density in the brain of patients with AD (5).

PATHOGENESIS An overwhelming body of evidence points to the fact that the Ab protein plays the central role in the pathogenesis of AD. Ab is derived from amyloid precursor protein (APP), a type I transmembrane protein encoded by the APP gene on chromosome 21 (6). APP is expressed in a variety of tissues, including neurons and glia, and contains a large extracellular domain, a single transmembrane domain, and a small cytoplasmic tail (Fig. 1). There are three main isoforms of APP derived by alternative splicing of the single APP gene. The two larger forms, APP751 and APP770, contain a 56-amino acid domain with homology to serine protease inhibitors (7). APP695 is the

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Figure 1 (A) Diagrammatic representation of amyloid precursor protein (APP695). (B) An enlarged view of amyloid b-protein (Ab). The a-, b-, and g-secretase cleavage sites are indicated by arrows and selected familial Alzheimer disease mutations shown below. Transgenic mice with the Swedish, London, and Indiana mutations and variations thereof have been used in some immunization studies.

shortest form, because it lacks this domain and is the most common form in neurons. The function of APP695 is not known, but in neurons it occurs mainly in synapses and seems likely to influence synaptic plasticity. Overexpression of APP suggests that it may have an anti-adhesion function. Mice lacking APP show only minor defects in neural function, perhaps due to compensation by an APP-like protein. APP is subject to extensive post-translational modification in the endoplasmic reticulum, the Golgi apparatus and the trans-Golgi network before being exported to the cell surface (8). APP is then further modified by proteolytic cleavage by a series of enzymes collectively termed secretases. Most APP is processed by cleavage with a-secretase releasing a soluble ectodomain sAPPa , into the extracellular space with retention of an intracellular C-terminal fragment (CTF). Processing via this pathway prevents the formation of full-length Ab (Fig. 1). By contrast, b-secretase [also known as memapsin 2 and as b-APP cleaving enzyme (BACE-1)] cleaves APP at a site immediately N-terminal to the Ab sequence, approximately 16 amino acids closer to the extracellular N-terminus of APP (9) (Fig. 1). In the nervous system, b-secretase activity is more prominent than in other tissues, thus favoring the production of Ab. Another key enzyme in the production of Ab is g-secretase, which cleaves APP within the transmembrane region at the C-terminus of Ab in a manner that releases soluble Ab from the cell (10). The exact cleavage site of g-secretase may vary slightly giving rise to peptides 40 to 43 amino acids in length. The most common species is Ab40, which is typically found in non-neuronal cells and tends to be more soluble than Ab42 and Ab43 (10). However, neuronal cells have a greater propensity to produce Ab42, which is a major protein species at the center of senile plaques. Even in the brain, however, 90% of Ab is Ab40, with the remainder being the less soluble and more fibrillogenic Ab42/43 (8). If the rate of production of Ab42 exceeds its rate of clearance, it may accumulate in the brain to cause neuronal toxicity. The basis of the toxic action of Ab is unknown, but there is a suggestion that secreted oligomers of Ab can inhibit hippocampal long-term potentiation, thus interfering with the

development of memory (11). In addition, the accumulation of Ab proto-fibrils on the cell surface may encourage the formation of free radicals that damage cell membranes and lead to neuronal death (12). Reactive inflammation also appears to contribute to the pathogenesis of AD. Although there is little evidence that inflammation triggers the development of AD per se, evidence from animal models suggests that certain inflammatory mediators, such as tumor necrosis factor a (TNF-a), prostaglandins and thromboxanes, are important drivers of the disease (13). The neurofibrillary tangles, which are the other hallmark histopathological lesion of AD, are made up mainly from the microtubule-associated protein tau, the normal function of which appears to be to stabilize microtubules. This function is regulated in the cell by the reversible phosphorylation and dephosphorylation of tau. The observation that tau in neurofibrillary tangles is excessively phosphorylated suggests that in affected neurons, the balance between the action of kinases and phosphatases is disturbed (14). In neurons, microtubules are involved in the axonal transport of proteins. The accumulation of abnormally phosphorylated tau may lead to destabilization of the cytoskeleton, neuronal dysfunction, and eventually to neuronal death. One possible underlying cause of the abnormal phosphorylation of tau in AD may be the presence of Ab itself. Interestingly, mutations in the tau gene lead to accumulation of aggregated tau (15). Patients with this type of disordered tau present with dementia, which, unlike AD, is characterized by frontal lobe atrophy and prominent behavioral abnormalities.

GENETICS Apart from age, the most significant risk factor for the development of AD is genetic. Familial AD, which accounts for fewer than 10% of all cases, is inherited in an autosomal dominant fashion. Apart from the fact that it generally occurs at a younger age, often less than 60, familial AD is indistinguishable, clinically and pathologically, from the

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sporadic form of the disease. Importantly, unraveling the genetic basis of familial AD has provided key insights into the molecular pathogenesis of AD in general, including strong support for the idea that Ab plays a central role in its pathogenesis. Early evidence for the role of chromosome 21 in the development of AD came from the observation that individuals with Down’s syndrome (trisomy 21) show identical pathological changes in the brain to those found in AD, and often develop this condition while still in their 30s (16). Intensive study of chromosome 21 revealed that some individuals with familial AD carry a point mutation within the APP gene (8,17). One of these mutations is the so-called Swedish mutation, a two-point mutation at amino acids 670 and 671 from Lys-Met to Asp-Leu. This change is upstream of the b-cleavage site (Fig. 1) and results in a five to eight-fold increase in the formation of both Ab40 and Ab42. A second mutation at amino acid 717, the ‘‘London’’ mutation, Val?Ile is adjacent to the g-cleavage site and specifically increases the production of Ab42. Although cases with these mutations account for less than 3% of all patients with early onset familial AD, the discovery of these mutations was vital in showing the clear link between abnormal APP processing and AD. Presumably, the association of Down’s syndrome with AD is due to overproduction of APP and consequently Ab (16). The largest proportion of patients with familial AD carry mutations in the presenilin gene (PS1) on chromosome 14 (8). The product of this gene is a large transmembrane protein that is an integral component of g-secretase (18). PS1 shares homology with a gene on chromosome 1 known as PS2 (19). One consequence of PS1 mutations is enhanced production of Ab42 suggesting that these mutations influence the activity of g-secretase. Thus, collectively, most early onset familial forms of AD are explained by excessive production of Ab42 either as a result of missence mutations in APP at or near the cleavage site of b- or g-secretase or by abnormal g-secretase activity per se. Despite the persuasive evidence incriminating Ab metabolism as the key abnormality in familial forms of AD, the molecular pathogenesis of sporadic, late onset of AD is poorly understood. The age of onset of AD is linked with the inheritance of particular isoforms of apo-lipoprotein E (ApoE) (20). ApoE is a protein that is involved in cholesterol metabolism transport and storage. The gene for ApoE is located on chromosome 19 and occurs as three common allelic variants termed ApoE2, E3, and E4, the products of which differ by only one amino acid. Individuals with the ApoE4 allele are significantly more likely to develop AD at an earlier age than those without it, with ApoE4 homozygous individuals at greatest risk (20). Although there is no evidence to suggest that ApoE4 is directly responsible for the development of AD, it may modify expression of the disease by involvement in Ab deposition or clearance.

ANIMAL MODELS Because much of the research which led to the serendipitous possibility of prophylactic and therapeutic immunization for AD has come from animal models of this disease, it is useful to consider some of these models. Before the identification of the genetic mutations associated with familial AD, animal models were mainly developed to reproduce the nonspecific neuropathology of AD, such as neuronal damage. Although naturally aging dogs may serve as a useful model of AD for some purposes (21,22), the discovery of the genetic basis of familial

AD permitted the generation of transgenic mice, which display various features of AD in humans (8,17,23). Early attempts to develop transgenic AD mice involved transfection with the normal human APP gene either in its entirety or as a C-terminal fragment. However, these animals failed to develop plaques presumably because they produced APP at approximately endogenous concentrations. Subsequent work showed that a key factor in plaque development is an elevated concentration of Ab42. This can be achieved through the use of promoters, such as platelet-derived growth factor (PDGF), prion protein (PrP), or Thy-1, to drive higher levels of protein expression (8). High concentrations of Ab are also obtained when APP with the Indiana, London, or Swedish mutations are used for transfection instead of the wild-type gene. The first successful mouse model of AD was reported in 1995 by Games et al. and termed PDAPP (24). This model is based on a complex genetic construct consisting of the promoter for the b-chain of PDGF and a mini-gene containing cDNA of portions of APP introns 6 to 8 bearing a V717F mutation in APP. PDAPP mice contain approximately 40 copies of the transgene, and produce human APP mRNA at levels significantly greater than endogenous App transcripts and levels of APP approximately ten times greater than endogenous mouse APP. At eight months of age, these animals develop deposits of Ab in the hippocampus, corpus callosum, and cerebral cortex. Although they suffer no significant loss of neurons, they do show impaired ability to perform novel ‘‘spatial’’ memory tasks in an age-dependent manner that correlates with the accumulation of Ab deposits (25). In 1996, a second mouse model, termed Tg2576, was developed. These animals express the 695 amino acid form of human APP carrying the Swedish mutation (K670N/M671L) under the transcriptional control of the hamster PrP promoter (26). The levels of APP produced by these mice are approximately five to six times higher than endogenous App, and they develop Ab deposits at 9 to 11 months of age. Tg2576 mice also show disordered behavior and memory in tasks involving different types of mazes. Another cDNA transgenic model that has been used in vaccine studies is termed TgCRND8 (27). These mice carry full-length APP695 cDNA with both the K670N/M671L and V717F mutations under the control of hamster PrP. In these animals, the APP transgene is expressed at levels five times greater than endogenous App, and Ab deposits occur as early as three months of age. The development of these and several other varieties of transgenic mice, which express abnormal APP, has established the relationship between APP, Ab production, and disorders of memory. Interestingly, however, although some of these transgenic mice show modest loss of neurons and synapses, none of the APP mutant mouse strains develop full-blown neurofibrillary tangles or show the pronounced neurodegeneration that is found in patients with AD (8). By contrast, transgenic mice, which express both abnormal APP and a mutant tau protein exhibit substantially enhanced neurofibrillary tangle pathology in the limbic system and olfactory cortex (28). This observation supports the hypothesis that a similar interaction occurs between Ab and tau in patients with AD.

IMMUNIZATION STUDIES IN ANIMALS The first steps to indicate that AD may be amenable to prevention or even treatment by immunization came in 1997 from a report by Solomon et al. (29) that antibodies raised against the

Chapter 87: Vaccines Against Alzheimer’s and Other Neurodegenerative Diseases

N-terminal region (1–28) of Ab bind to in vitro–formed bamyloid assemblies, leading to disaggregation of the fibrils and partial restoration of the peptide’s solubility. In addition, some monoclonal antibodies raised against soluble b-peptide (1–28) prevented fibrillar aggregation of Ab in vitro, suggesting that site-directed antibodies can interfere with the aggregation of Ab and trigger its reversal to nontoxic, normal components. This work was extended by Schenk et al. of Elan Pharmaceuticals who immunized PDAPP transgenic mice 11 times with 100 mg of a synthetic peptide preparation of human Ab42 together with Freund’s adjuvant (30). Control mice received either serum amyloid peptide (SAP) or phosphate buffered saline (PBS), or were left untreated. Mice given Ab42 developed and maintained high titers of serum antibody to this peptide. These antibodies cross-reacted with mouse Ab without causing obvious tissue damage. One group of mice in this study was immunized over a period of 11 months starting at six weeks of age, that is, before the development of amyloid plaques. These mice were killed at age 13 months, and their brains were compared with those of the three control groups. The results showed that mice immunized with Ab42 had significantly less (P  0.001) Ab burden (0%, as measured by quantitative image analysis) compared with the groups which received saline (2.2%), SAP (5.7%), or no treatment (2.7%). In addition, the presence of dystrophic neurites was reduced from 0.3% in controls to 0% in immunized animals; and immunoreactivity for glial fibrillary acidic protein, which is a marker of Ab plaque-associated gliosis, was reduced from 6% in control mice to 1.6% in those given Ab42. This study indicates that immunization with Ab42 can prevent the development of AD-like neuropathological lesions in PDAPP mice. However, it was unclear if immunization could influence the course of the disease if plaques were present when immunization was started. To address this important issue, Schenk et al. (30) immunized some PDAPP mice that were 11 months old, an age when numerous Ab plaques are already present. Half the mice were killed after four months of treatment, and the rest three months later. The results were that immunized mice showed a 96% reduction in plaques after four months’ treatment, and greater than 99% reduction after seven months, compared with controls (P < 0.001). Moreover, the Ab burden in the seven-month treatment group was 0.01% compared with 4.9% in control animals given PBS. Similar findings have been reported by other workers using DgCRND8 and Tg2576 mice (27,31). Despite these encouraging results, it was not clear if the reduction in pathological changes due to the immunization would result in improvements in cognitive function. This question was addressed by Morgan et al. (31), who used a combination of a swim maze and radial arm maze to evaluate cognitive defects in Tg2576 and PS1N146L double transgenic mice. These mice show high levels of Ab production and plaque development by 12 weeks of age, which increases nearly 200-fold by one year. The memory task used by Morgan et al. requires mice to learn the spatial location of a submerged escape platform on the basis of visual clues, and to navigate the platform from different starting points (31). Transgenic and control mice were immunized with either Ab42 or a placebo protein, keyhole limpet hemocyanin (KLH). Normal mice showed no change in cognitive function after immunization with either protein, demonstrating that immunization with Ab42 has no overt deleterious effect on these animals. As expected, transgenic mice immunized with KLH at 11.5 months showed virtually no learning ability four months later, but the Ab immunized transgenic mice showed cognitive performance

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similar to that of non-transgenic animals. Ab-immunized mice also demonstrated a partial reduction in amyloid burden at the end of the study. Similar findings were obtained by Janus et al. (27) in TgCRND8 mice (APP695K670N/M671L and V717F), which normally develop increased soluble Ab, amyloid plaques and deficits in spatial learning at three months. In this study, age- and sex-matched transgenic mice and non-transgenic littermates were immunized at 6, 8, 12, 16, and 20 weeks of age with either Ab42 or a control protein, islet-associated polypeptide, as b-pleated sheets. Through the use of the more conventional water maze than that used by Morgan et al., these investigators showed that repeated vaccination of TgCRND8 mice with Ab42 reduced cognitive dysfunction concurrently with reduced cerebral fibrillar Ab compared with control mice, although total levels of Ab in the brain were not affected. On the basis of these somewhat unexpected results, Janus et al. concluded that either an approximately 50% reduction in dense-core Ab plaques is sufficient to improve cognition or immunization may modulate the activity or abundance of a small subpopulation of especially toxic Ab species. Analysis of the immune response of PS1 transgenic mice to immunization with Ab42 in complete Freund’s adjuvant revealed that specific antibodies are detectable only after the third boost (32), with highest antibody responses directed against the N-terminus of Ab. After the sixth dose, antibody titers tend to plateau and thereafter are maintained above the half-maximal titer for at least five months without further boosting. Assays of specific immunoglobulin isotypes in mice immunized with Ab have demonstrated a preponderance of specific IgG 1 and IgG 2B , indicating a T h 2-type immune response (32). The major contribution of antibodies to the amyloid depleting effects of Ab immunization has been shown in a series of studies involving passive immunization with anti-Ab antibodies. Bard et al. (33) treated 8-to 12-month old PDAPP mice for five months with weekly injections of murine monoclonal or polyclonal antibodies directed against Ab, and reduced plaque burden by more than 80% with a corresponding reduction in cortical levels of Ab42. The key role of antibodies was confirmed by the finding that T cells from passively immunized mice showed no proliferative response when stimulated with Ab, indicating that plaque clearance does not require T-cell immunity. Subsequent studies showed that (i) only antibodies that recognized the N-terminal region of Ab mediated plaque clearance; (ii) plaque binding correlated with the clearance response and neuronal protection, (iii) IgG2a effected plaque clearance and neuronal protection more than other isotypes; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Ab itself; and (v) complement activation was not required for plaque clearance (34). An important finding of these studies was that contrary to expectations, antibodies that had been administered peripherally entered the central nervous system and bound to amyloid plaques (33). An ex vivo assay with sections of brain tissue from PDAPP mice or patients with AD showed that antibodies to Ab can trigger microglial cells to clear plaque through Fc receptor-mediated phagocytosis with subsequent peptide disaggregation. A separate study showed that the direct application of anti-Ab antibodies to the surface of the brain of living PDAPP mice can reduce Ab deposits in the immediate vicinity of the application (35). The unexpected finding that peripherally administered antibodies can traverse the blood-brain barrier may be explained by the fact that Ab

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itself is transported across this barrier by a receptor-mediated pathway, and that antibodies bound to Ab may be able to utilize the same pathway (36). For passive immunization to succeed in humans, therefore, there may need to be a critical concentration of circulating Ab, which generally is far lower than that in transgenic mice (37). Another potential problem with passive immunization of humans against AD is that Ab peptides within the plaques of transgenic mice are physically and chemically distinct from those found in AD, with the latter often displaying a degraded N-terminus, extensive posttranslational modifications, and a reduced tendency toward disaggregation (38). On the other hand, some studies suggest that the major targets of anti-Ab antibodies are not plaques per se but soluble toxic oligomers of Ab (11,39,40). This observation provides an explanation for the finding that the cognitive function of TgCRND8 mice immunized with Ab showed significant improvements without a major reduction in plaque burden (27). Anti-Ab antibodies can also reverse cholinergic dysfunction, which contributes to memory impairment in AD (41). An alternative view of how peripherally administered antibodies may be beneficial in mouse models of AD has been provided by DeMattos et al. (42) who administered an anti-Ab monoclonal antibody intravenously to PDAPP mice and showed that this led to a 1000-fold increase in plasma levels of Ab. Because levels of Ab in plasma of untreated animals are low, DeMattos et al. proposed that antibodies in the plasma act as a ‘‘peripheral sink,’’ which promotes the efflux of Ab from the brain to the plasma. These authors went on to show that long-term administration of antibody significantly reduced Ab burden in the brain without the antibody crossing the bloodbrain barrier or binding to intracerebral Ab. The reasons for the differences between the findings of Bard et al. (33) and DeMattos et al. (42) regarding the transport of anti-Ab antibodies across the blood-brain barrier are not clear, but there is no doubt that peripherally administered antibodies can deplete Ab in the brain of transgenic mice (43). Whether such treatment will be effective in humans with AD is unknown. If the principal target of anti-Ab is soluble Ab, active or passive immunization of patients with established AD may be less effective than vaccinating individuals with early disease. Accordingly, effective implementation of a vaccine for AD would necessitate large-scale screening of the general population to identify at-risk individuals who could benefit from immunization. Because further studies of Ab vaccine efficacy would need to be undertaken in humans, several investigators have examined modes of vaccine delivery that do not require Freund’s adjuvant, which is not licensed for use in humans. Some of the alternative approaches to vaccine delivery that have been successfully trialed in animal models include intranasal and transcutaneous immunization with various from of Ab (44–46), immunization with Ab DNA or Ab1-9 on virus-like particles (47–49), and the administration of Ab42 in biodegradable poly(lactide-co-glycolide) microspheres (50) or as Ab1-15 dendrimers (51). In an attempt to reduce the potential toxicity of Ab42, which can cross the blood-brain barrier to form toxic fibrils and possibly seed new fibril formation, Sigurdsson et al. (52) used nontoxic-non-amyloidogenic Ab homologous peptide (K6Ab1– 30-NH2) to immunize Tg2576 mice. This peptide retains the two major immunogenic sites of intact Ab, which are contained within residues 1 to 11 and 22 to 28 of Ab42. This study showed

that administration of this compound for seven months reduced cortical and hippocampal brain amyloid burden by between 80% and 90%, and reduced brain levels of soluble Ab42 by 57% (52). An even more reductionist approach to immunization has been adopted by Frenkel et al. (53–55), who immunized guinea pigs with a genetically engineered filamentous phage displaying the epitope, EFRH, which corresponds to amino acids 3-6 within Ab (Fig. 1). As this region of Ab controls the formation and disaggregation of Ab fibrils, binding of antibodies to this epitope prevents self-aggregation of Ab as well as enabling the re-solubilization of preformed aggregates (29). Apart from lower toxicity, the phage delivery system also has the advantage of being highly immunogenic, thus obviating the need for separate adjuvants. The use of a phage delivery system may also overcome the immune hyporesponsiveness to Ab found in certain strains of transgenic mice and could contribute to the pathogenesis of AD while simultaneously reducing the potential efficacy of future immunization strategies based on Ab (56).

IMMUNIZATION OF HUMANS Although none of the animal models investigated to date has shown complete remission or prevention of disease occurrence, the encouraging results obtained with Ab immunization of transgenic mice led to fast-tracking of human trials of Ab immunization (57). A phase I/II trial to examine the safety of a synthetic Ab42 vaccine, known as AN-1792, administered as a single dose to patients with moderate AD was commenced in December 1999, and showed the vaccine to be well tolerated. In early 2002, however, reports emerged of a severe reaction resembling experimental allergic meningoencephalitis in 18 of 300 recipients of AN-1792 (58). Importantly, however, postmortem analysis on some participants in the trial demonstrated the active removal of Ab deposits through the action of microglia and macrophages engulfing aggregated Ab (59,60). While all clinical trials with this material were temporarily suspended (59), new trials have progressed through phase II using other modalities of Ab immunization. Clearly, the early setback will necessitate improved understanding of vaccine action and toxicity. Possible explanations for the side effects of AN-1792 include (i) toxicity directly attributable to the peripheral administration of Ab42, (ii) the induction of T cell–mediated autoimmunity, (iii) the capacity for Ab antibodies to increase levels of soluble amyloid leading to cerebrovascular toxicity (61), and (iv) the development of antibodies to APP and Ab peptides, which are normal constituents of brain tissue. However, although antibodies elicited by AN-1792 recognized b-amyloid plaque, diffuse Ab deposits, and vascular amyloid in blood vessels, they did not cross-react with full-length APP or soluble Ab42 (62). Another explanation for antibody-mediated damage comes from a report by Pfeifer et al. (63), who showed that reduction in diffuse amyloid in transgenic mice given passive anti-Ab immunotherapy was accompanied by cerebral microhemorrhages. The setback experienced in early human trials of AD vaccines does not spell the end of immunization against AD, especially in the light of reports that recipients of the AN-1792 vaccine may have experienced some benefit in terms of slowed cognitive decline and higher neuropsychological test battery scores compared with recipients of placebo (64,65). One major consequence of the experience with AN-1792, however, will be an increased need to address the safety of future vaccine

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candidates. Strategies to avoid vaccine-induced encephalitis could include the construction of vaccines that lack the T-cell epitopes, which trigger adverse reactions, or that target novel epitopes on abnormal forms of Ab (66). The design of such second-generation vaccines would be facilitated by an improved understanding of the pathogenesis of AD, including the physiological roles of APP and Ab and the consequences of artificially induced anti-Ab immunity (67). Alternative approaches to immunization could involve entirely novel vaccines, such as b-secretase, which has been shown to reduce Ab deposits and improve cognitive function in Tg2576 mice in the absence of an inflammatory response (68). The increasing prevalence of AD, its potentially enormous costs, and the promise of its prevention or arrest through immunological interventions, ensures that the search for an effective AD vaccine will continue. Indeed, the current levels of activity in this area of AD research have stimulated similar investigations of immune modulation of aggregated proteins in other neurodegenerative diseases, such as Parkinson’s disease, frontotemporal tauopathies, CJD, and amyotrophic lateral sclerosis. Initial preclinical experiments have shown encouraging effects, leading to the revolutionary idea that a wide range of common neurodegenerative diseases may be amenable to therapeutic interventions of this type.

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reduces Alzheimer’s disease-associated pathology in transgenic mice. Am J Pathol 2001; 159:439–447. Frenkel D, Katz O, Solomon B. Immunization against Alzheimer’s b-amyloid plaques via EFRH phage administration. Proc Natl Acad Sci U S A 2000; 97:11455–11459. Frenkel D, Dewachter I, Van LF, et al. Reduction of b-amyloid plaques in brain of transgenic mouse model of Alzheimer’s disease by EFRH-phage immunization. Vaccine 2003; 21:1060–1065. Frenkel D, Dori M, Solomon B. Generation of anti-b-amyloid antibodies via phage display technology. Vaccine 2004; 22:2505–2508. Monsonego A, Maron R, Zota V, et al. Immune hyporesponsiveness to amyloid b-peptide in amyloid precursor protein transgenic mice: implications for the pathogenesis and treatment of Alzheimer’s disease. Proc Natl Acad Sci U S A 2001; 98:10273–10278. Thatte U. AN-1792 (Elan). Curr Opin Investig Drugs 2001; 2:663–667. Check E. Nerve inflammation halts trial for Alzheimer’s drug. Nature 2002; 415:462. Masliah E, Hansen L, Adame A, et al. Ab vaccination effects on plaque pathology in the absence of encephalitis in Alzheimer disease. Neurology 2005; 64:129–131. Nicoll JA, Wilkinson D, Holmes C, et al. Neuropathology of human Alzheimer disease after immunization with amyloid-b peptide: a case report. Nat Med 2003; 9:448–452. Patton RL, Kalback WM, Esh CL, et al. Amyloid-b peptide remnants in AN-1792-immunized Alzheimer’s disease patients: a biochemical analysis. Am J Pathol 2006; 169:1048–1063. Hock C, Konietzko U, Papassotiropoulos A, et al. Generation of antibodies specific for b-amyloid by vaccination of patients with Alzheimer disease. Nat Med 2002; 8:1270–1275. Pfeifer M, Boncristiano S, Bondolfi L, et al. Cerebral hemorrhage after passive anti-Ab immunotherapy. Science 2002; 298:1379. Gilman S, Koller M, Black RS, et al. Clinical effects of Ab immunization (AN1792) in patients with AD in an interrupted trial. Neurology 2005; 64:1553–1562. Hock C, Konietzko U, Streffer JR, et al. Antibodies against bamyloid slow cognitive decline in Alzheimer’s disease. Neuron 2003; 38:547–554. Wang CY, Finstad CL, Walfield AM, et al. Site-specific UBITh amyloid-b vaccine for immunotherapy of Alzheimer’s disease. Vaccine 2007; 25:3041–3052. Weiner HL, Frenkel D. Immunology and immunotherapy of Alzheimer’s disease. Nat Rev Immunol 2006; 6:404–416. Chang WP, Downs D, Huang XP, et al. Amyloid-beta reduction by memapsin 2 (beta-secretase) immunization. FASEB J 2007; 21: 3184–3196.

88 Vaccination for Autoimmune and Other Chronic Inflammatory Disorders Leonard C. Harrison Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, Burnet Clinical Research Unit, Parkville, Victoria, The Royal Melbourne Hospital, Parkville, Victoria, Australia

INTRODUCTION At the dawn of the new millennium, chronic noncommunicable disorders are the major cause of morbidity and mortality among populations living in industrialized and transitional countries. The rising tide of autoimmune and allergic diseases is overshadowed even by diseases associated with subtle lowgrade inflammation including obesity, insulin resistance and type 2 diabetes (T2D), atherosclerosis and cardiovascular disease, and, arguably, Alzheimer’s disease and cancer. These disorders demand novel preventative and therapeutic approaches, one of which is vaccination. Classically, after Jenner, vaccination has been a means of inducing an immune response resulting in resistance to infectious disease. This ‘‘positive’’ vaccination is targeted against exogenous, nonself antigens. Vaccination for therapeutic purposes can also be targeted to endogenous self-antigens, to achieve gain or loss of function depending on the type of induced immune response. Like many drugs, antibodies to receptors or other molecules can have agonist or antagonist properties and thereby modify cell function. This is illustrated by autoantibodies in experiments of nature, for example, by agonist autoantibodies to the thyrotropin receptor that cause hyperthyroidism in Graves’ disease or by antagonist autoantibodies to the acetylcholine receptor that cause muscle weakness in myasthenia gravis. Just as experimental immunologists employ antibodies passively in vitro and in animal models to block mediators of pathology, so also can vaccination be used to induce antibodies that modify cell function in vivo. Some understanding of physiology and disease mechanisms and a glance at the index of a medical textbook would suggest a range of applications for ‘‘autovaccination,’’ from suppression of inflammation to prevention of conception. The author is not aware of autovaccination being used to deliberately up-regulate ‘‘self’’ function but there is no theoretical reason why the immune system could not be manipulated to do so. Likewise, the cellular arm of the adaptive immune system can be manipulated to modify the function of the immune and other systems. T cells that recognize self-antigen peptides presented by antigen-presenting cells in tissues or draining lymph nodes can modify other cells and the local environment, for example, induce cell death, suppress antigen-presenting cell or pathogenic T-cell function, or reduce inflammation and vascular permeability. This is illustrated by CD8þ cytotoxic T-lymphocyte (CTL) immunity against tumors on the one

hand and by Tregs that protect against autoimmune disease on the other. Vaccination strategies to prevent or ameliorate autoimmune disease could: (i) avert causative environmental agents, (ii) delete or inactivate pathogenic T cells, (iii) induce protective/ regulatory T cells or therapeutic antibodies, or (iv) promote a therapeutic effect downstream of autoimmune pathology [an example might be to enhance insulin sensitivity in autoimmune or type 1 diabetes (T1D)]. Vaccination with an autoantigen to induce, paradoxically, disease-specific immune tolerance/ protection can be termed ‘‘negative vaccination.’’ It is based on the concept that autoantigen-specific immune tolerance mechanisms are physiological and can be boosted or restored to prevent pathological autoimmunity (1). For autoimmune disease, autoantigen-specific vaccination is the therapeutic Holy Grail. It is efficacious in rodent models but has yet to be effectively translated to humans. The likely reasons for this are discussed later. In theory, vaccination with autoantigen should be relatively safe and inherently more acceptable for prevention of autoimmune disease in asymptomatic individuals than treatment with conventional, nonspecific immunosuppressive agents. However, allergic reactions and acceleration rather than retardation of underlying disease are possible outcomes that require attention. In particular, vaccination with autoantigen is a double-edged sword that can also induce CTLs, as exemplified by attempts to induce antitumor immunity. In the context of autoimmune disease, the desired outcome, immune tolerance/protective immunity, depends on a range of factors. These include the ‘‘load’’ of activated, pathogenic effector cells to overcome, route of delivery (e.g., mucosal vs. systemic), nature of the autoantigen (e.g., the presence of CTL epitopes), dose of autoantigen, and context of autoantigen recognition (e.g., the nature of the antigen-presenting cell, cytokine milieu). Protective immunity has been achieved in rodent models by administering autoantigen protein, peptide, or DNA via ‘‘tolerogenic’’ routes, cell types, modes, or forms (Table 1), to delete or anergize pathogenic lymphocytes and/or induce regulatory T cells (Tregs) (2). Some Tregs secrete the immunosuppressive cytokines IL-10 or TGF-b that suppress the ability of antigen-presenting cells to elicit effector T-cell responses to any antigen, a phenomenon called ‘‘bystander suppression.’’ Thus, although autoimmune disease is frequently associated with immunity to more than one autoantigen, bystander suppression by Tregs stimulated by one antigen

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Table 1 Autoantigen-Specific Vaccination Strategies That Prevent Experimental Autoimmune Disease Administration of autoantigen: . Via a tolerizing route . Mucosal . Dermal . Via a tolerizing cell type . Immature dendritic cell . Via a tolerizing mode . With blockade of costimulator ‘‘second signal’’ . In a tolerogenic form . Soluble IV or IP . Soluble peptide-MHC IV . As ‘‘altered peptide ligand’’ . As aggregated Ig chimera Abbreviations: IV, intravenous; IP, intraperitoneal; MHC, major histocompatibility complex.

locally at the site of the lesion or in the draining lymph nodes affects T-cell responses to all autoantigens and obviates knowing if the autoantigen that induced Tregs is the primary driver of pathology. This chapter deals with vaccination against self, mainly for the prevention of autoimmune disease. Although other

Figure 1 Autoantigen vaccination in rodent models of autoimmune disease. Abbreviations: ADM, autoimmune diabetes mellitus; EAA, experimental autoimmune arthritis; EAE, experimental autoimmune encephalomyelitis; EAMG, experimental autoimmune myasthenia gravis; EAN, experimental autoimmune neuritis; EAT, experimental autoimmune thyroiditis; EAU, experimental autoimmune uveitis; AchR, acetylcholine receptor; GAD, glutamic acid decarboxylase; HSP, heat shock protein; IRBP, interphotoreceptor retinoid binding protein; MBP, myelin basic protein; PNM, peripheral nerve myelin; Tg, thyroglobulin.

examples are discussed, it draws heavily on T1D for three objective reasons: (i) the ability to identify humans at risk and to predict their risk many years before the clinical onset of disease, which makes T1D paradigmatic for prevention of autoimmune disease; (ii) proof-of-concept for autoantigen-specific vaccination with whole protein, peptide epitopes or DNA is firmly established in the nonobese diabetic (NOD) mouse, uniquely a spontaneous model of autoimmune disease but illustrative of other rodent models of induced autoimmune disease such as experimental allergic encephalomyelitis (EAE), collagen-induced arthritis, uveitis, and myastenia gravis (2) (Fig. 1), and (iii), following from (ii), trials of islet autoantigen-specific vaccination have been completed or are ongoing in humans at risk for T1D. Also, the author concedes that his expertise is mainly in T1D. Vaccination for noninfectious disease is a burgeoning area covering an expanding array of disorders and approaches that cannot all be reviewed here for lack of space. Cancer and allergy are dealt with in other chapters. The author apologizes to the many investigators whose work has not been cited.

TYPE 1 DIABETES Introduction T1D is an autoimmune disease in which genes and environment interact to effect cell-mediated immune destruction of insulin-producing b cells in the islets of the pancreas. In most cases, a subclinical phase of b-cell pathology lasting many months or years precedes significant insulin deficiency leading to symptomatic hyperglycemia (Fig. 2). Primary prevention of T1D, before initiation of the disease process, might be achieved by classical ‘‘positive’’ vaccination of the community against an infectious agent that triggered or promoted disease, if one could be identified. It is conceivable that some cases of T1D may have already been prevented by vaccination—to rubella, since strong circumstantial evidence suggests that congenital rubella was associated with development of T1D (3,4). Epidemiological evidence has implicated coxsackie and echoviruses (5,6) and rotaviruses (7). It will be of interest to see if recently introduced vaccines for rotavirus alter the prevalence of T1D in genetically at-risk populations. If ubiquitous viruses have a role, it is likely to be complex. In the NOD mouse, diabetes is accelerated in a germ-free environment (8) and the ‘‘hygiene hypothesis’’ posits that cleaner living conditions and/or reduced infectious exposure are responsible for the increasing incidence of autoimmune and allergic disorders (9,10). Our studies in the NOD mouse suggest that the timing and context of rotavirus infection is critical in determining if diabetes is accelerated or retarded (11). A ‘‘negative vaccine,’’ provided it was both efficacious and safe could also be used for primary prevention, at least in children with a high genetic risk. After disease initiation, which occurs at an indeterminate point in time, the subclinical phase before diagnosis of T1D is a window of opportunity for secondary prevention, when autoantigen-specific vaccination would be a preferred approach because of relative safety. In this phase, at-risk individuals with underlying islet inflammation are identified by the presence of circulating autoantibodies to islet antigens: islet cells antibodies (ICA) revealed by indirect immunofluorescence on pancreas sections and specifically antibodies to (pro)insulin, the molecular weight 65,000 isoform of glutamic acid decarboxylase (GAD) 65, tyrosine phosphatase– like insulinoma antigen 2 (IA2), and b-cell-specific zinc

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Figure 2 Natural history of type 1 diabetes.

transporter 8 (12,13) (Fig. 2). Many different types of conventional immune suppressive/modifying agents have been used over the past 25 years in attempts, largely unsuccessful in the longer term, to retard loss of residual b-cell function in people with recent-onset T1D (14,15). It is unlikely that, used alone, autoantigen-specific vaccination would be effective in late preclinical or recent-onset clinical disease, but conceivably it may be synergistic then in combination with nonspecific immune suppressive/modifying agents that reduce the burden of pathogenic immunity, with the potential for b-cell recovery and possible regeneration.

Vaccination in the Nonobese Diabetic Mouse A prerequisite for most human therapeutics is the demonstration of efficacy and safety in animal models. The NOD mouse has contributed greatly to our understanding of autoimmune diabetes and to the view that autoantigen-specific vaccination can prevent T1D (16,17). NOD mice and humans with T1D share a number of important features: genetic susceptibility dominated by the major histocompatibility complex (MHC) but a substantial effect of environment on genetic penetrance, a prolonged subclinical phase, autoimmunity to (pro)insulin and GAD65, and disease transmission via bone marrow. Studies in the NOD mouse, as well as in the biobreeding (BB) rat spontaneous model of T1D, demonstrate that CD8 T cells are the ultimate cellular effectors of b-cell death. Several lines of evidence demonstrate directly in the NOD mouse and indirectly in children with T1D that (pro)insulin is the preeminent autoantigen driving b-cell destruction (18,19). The NOD mouse has been criticized because it is inbred and lives in a controlled specific pathogen-free (SPF) environment and its diabetes incidence is reduced by many interventions (17). However, very

few interventions actually prevent diabetes in the NOD mouse, most simply delay diabetes onset, others have no effect and therefore are not reported, none are really effective in the late subclinical phase, and most have not been tested in humans. Mucosal Vaccination Oral tolerance as a treatment for autoimmune disease in animals and humans has been comprehensively reviewed (20). Many investigators have shown that NOD mice can be partially protected from spontaneous diabetes by mucosal administration of islet autoantigen, (pro)insulin or GAD65 proteins or peptides (summarized in Table 2). DNA has also been used for vaccination. Compared with protein, antigen encoded as DNA has potential advantages as a therapeutic: ease of handling, stability, purity (less risk of contaminants), production of native protein (nature does the work) with no requirement for protein purification, and sustained delivery (less frequent dosing). However, a potential drawback of DNA is that like viral nucleic acid, it may elicit strong CD8þ CTL responses to encoded MHC class I-restricted epitopes. CTL induction is not however unique to DNA, and the potential danger of generating pathogenic CTLs after mucosal administration of protein, in addition to Tregs, is recognized. For example, in C57BL/6 mice, we found that oral, aerosol, or nasal ovalbumin (OVA) protein induced both tolerance and pathogenic CTLs (41), although blockade of CD40 ligand prevented priming of CTLs while sparing tolerance (42). Intranasal vaccination of NOD mice with plasmid DNA encoding mouse (pro)insulin II induced CD4þ Tregs that suppressed adoptive transfer of diabetes with ‘‘diabetogenic’’ spleen cells, but diabetes was not prevented in the treated mice unless DNA vaccination was performed under cover of CD40 ligand blockade (43).

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Table 2 NOD Mouse Studies of Mucosal Vaccination with Islet Autoantigen Study Result

References

Oral porcine insulin reduces diabetes incidence Human insulin induces CD4 Tregs that transfer protection to naı¨ve mice Protection following oral insulin is associated with decreased expression of IFN-g-secreting Th1 T cells in pancreas and pancreatic lymph nodes Oral insulin-induced CD4 Tregs prevent immune-mediated diabetes induced by LCMV infection of mice expressing the viral nucleoprotein of LCMV under control of the rat insulin promoter in b cells. Protective effect of oral insulin is enhanced by simultaneous feeding with IL-10, bacterial component OM-89 or Schistosome egg antigen, all of which promote Th2 responses. Fusion of insulin to CTB significantly improves efficacy of oral insulin. Feeding potatoes transgenically expressing CTB-insulin fusion proteins reduces diabetes incidence. Oral recombinant vaccinia virus encoding CTB-insulin or CTB-GAD65 fusion proteins reduces diabetes incidence. Oral GAD reduces diabetes incidence. Oral insulin, insulin B chain, or GAD peptide administered to neonates reduce diabetes incidence. This suggests that even in very young infants, presentation of autoantigen to the mucosa (e.g., insulin in milk) could be prophylactic. Aerosol insulin at eight weeks, after the onset of subclinical disease, induces CD8 gd Tregs and reduces diabetes incidence. Naso-respiratory administration is direct and minimizes antigen degradation. Intranasal insulin B chain peptide (aa9-23), an epitope recognized by islet-infiltrating CD4 T-cell clones capable of adoptively transferring diabetes to naı¨ve mice, induces CD4 Tregs and protects NOD mice from diabetes. Intranasal proinsulin B-C chain peptide, a T-cell epitope in humans at risk for T1D, induces CD4 Tregs; deletion of amino acids for CD8 T-cell recognition improves efficacy for diabetes prevention. Intranasal T-cell epitope peptides from GAD65 induce CD4 Tregs and reduce diabetes incidence, and prolong syngeneic islet graft survival, i.e., recurrent autoimmune disease.

21 22 23,24 25 26–29 30 31 32 33 34

35 36 37,38 39,40

Abbreviations: LCMV, lymphocytic choriomeningitis virus; CTB, cholera toxin B subunit; Tregs, regulatory T cells; T1D, type 1 diabetes; GAD, glutamic acid decarboxylase.

Systemic Vaccination ‘‘Vaccination’’ with agents that stimulate innate immune pathways via toll-like and other pattern-recognition receptors, namely, complete Freund’s adjuvant, bacillus Calmette-Guerin (BCG), Schistosoma mansoni (44,45), and DNA or CpG oligonucleotide (46), reduced the incidence of diabetes in NOD mice but had no effect to preserve residual b-cell function in humans with T1D (Table 3). Systemic administration of islet autoantigens also protects NOD mice from diabetes (Table 4). Vaccination with stem cells engineered to encode autoantigen is a particularly effective way to achieve immune tolerance. Allogeneic or mixed allogeneic bone marrow transplantation has been used for severe autoimmune diseases but is unsuitable for prevention because of the requirement for cytotoxic conditioning of the recipient and the risk of graft rejection and graftversus-host disease (61). To avoid these, we used autologous hematopoietic stem cells (HSCs) or their resting, tolerogenic dendritic cell progeny to introduce autoantigen into the immature hematopoietic compartment. Transfer into young, irradiated NOD mice of 103 syngeneic HSCs encoding (pro) insulin expression in antigen-presenting cell progeny fully prevented diabetes (60). This effect appeared to depend on antigen expression by ‘‘resting’’ immature dendritic cells (62,63). Despite its appeal, this form of cell therapy faces scientific and safety-related hurdles in translation to humans, in particular how to introduce genes into stem cells without risk of oncogenesis and how to modify a mature immune system without exposure to toxic conditioning regimens. No doubt, these will be overcome with time. DNA Vaccination Compared with protein, antigen encoded as DNA has potential advantages as a therapeutic. These include ease of handling, stability, purity (less risk of contaminants), production of native protein (nature does the work) with no requirement for protein

purification and sustained delivery (less frequent dosing). Many reports attest to the variable efficacy of vaccination with DNA encoding autoantigen alone or with a cytokine or other immunoregulatory molecule, in mouse models of autoimmune disease, as reviewed for the NOD mouse (64). In the case of insulin (57,65), this has set the stage for human trials (see below). A potential drawback of DNA is that, like viral nucleic acid, it may elicit strong CD8þ CTL responses to encoded MHC class I-restricted epitopes. CTL induction is not, however, unique to DNA, and the potential danger of generating pathogenic CTLs after mucosal administration of protein, in addition to Tregs, is well recognized. For example, in C57BL/6 mice, we found that oral, aerosol, or nasal OVA protein induced both tolerance and pathogenic CTLs (41), although blockade of CD40 ligand prevented priming of CTLs while sparing tolerance (42). Intranasal vaccination of NOD mice with plasmid DNA encoding mouse (pro)insulin II induced CD4þ Tregs that suppressed adoptive transfer of diabetes with diabetogenic spleen cells, but diabetes was not prevented in the treated mice unless DNA vaccination was performed under cover of CD40 ligand blockade (43).

Vaccination Trials in Humans Randomized control trials (RCTs) of vaccination to prevent T1D in humans are summarized (Tables 3, 5, and 6). Several will be discussed in more detail to emphasize particular principles or outcomes. Two trials of oral insulin (up to 7.5 mg daily for 12 months) in recently diagnosed patients showed no protective effect on residual b-cell function (53,54). The multicenter Diabetes Prevention Trial (DPT)-1 was launched in the United States in 1994 to determine whether treatment with either systemic or oral insulin would delay or prevent the onset of diabetes in at-risk relatives. Previously, intensive systemic insulin therapy had been reported to prolong the ‘‘honeymoon’’

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Table 3 Vaccination Trialsa for Tertiary Prevention of T1D in Children or Adults with Recent-Onset T1D Trial

Subjects (n)

Follow-up (mo)

Intradermal BCG

26

18

Intradermal BCG

94

24

Q fever antigen

39

12

Heat shock protein 60 p277 peptide (‘‘DiaPep’’)

35

10

Parenteral insulin (IV vs. SC 2 wk) Subcutaneous insulin

19

12

10

Outcome

References

No effect on glucagon-stimulated C-peptide, insulin dose or HbA1C No effect on mixed meal-stimulated C-peptide, insulin dose or HbA1C No effect on glucagon-stimulated C-peptide or insulin dose Decrease in glucagon-stimulated C-peptide and insulin dose in placebo but not treated group. Higher meal and glucagon-stimulated C-peptide and lower HbA1c Higher C-peptide response to oral glucose, HbA1C unchanged Higher basal and glucagon-stimulated C-peptide, more remissions No effect on basal C-peptide, HbAIC, insulin dose or insulin antibodies No effect on basal, glucagon-or mealstimulated C-peptide, HbAIC, insulin dose or islet antibody levels Borderline reduction in loss of fasting and meal-stimulated C-peptide; no effect on HbA1c or insulin requirement.

47 48 Schmidli R not published 49

50 51

Subcutaneous insulin and sulphonylurea (Glipizide) Oral insulin

27

12

52

80

12

Oral insulin

131

12

Subcutaneous GAD65-alum (2x 20mg) (DiamydTM)

70

30

Nasal insulin (Melbourne Intranasal Insulin Trial III): 1.6 mg insulin or placebo daily 10 days, then weekly for year. Intramuscular plasmid DNA encoding proinsulin (BHT3021): 0.3, 1, 3 or 6 mg weekly for 12 wk. Parenteral insulin B chain in incomplete Freund’s adjuvant Parenteral insulin B chain 9–23 ‘‘altered peptide ligand’’ NBI6024-0101 (‘‘Neurocrine’’)

60

24

Leonard C. Harrison, Royal Melbourne Hospital, Victoria, Australia

54

12

Bayhill Therapeutics, California, U.S.A.

12

24

Ongoing

Orban T, in progress

188

25

Ongoing

Gottlieb PA, in progress

53 54

55

a Trials that have been designed but not yet opened or are incomplete or not reported are shown in italics. Abbreviations: BCG, bacillus Calmette-Guerin; T1D, type 1 diabetes; GAD, glutamic acid decarboxylase; IV, intravenous; SC, subcutaneous.

Table 4 NOD Mouse Studies of Systemic Vaccination with Islet Autoantigen Study Result

References

Subcutaneous or intravenous GAD65 in incomplete Freund’s adjuvant at 8 or 12 wk of age, after onset of islet inflammation, induces Tregs and reduces diabetes incidence. Intramuscular plasmid DNA encoding insulin B chain induces IL-4-secreting Tregs and protects mice expressing LCMV nucleoprotein transgene in b cells from diabetes after LCMV infection. Intramuscular plasmid DNA encoding GAD65-IgGFc and IL-4 prevents diabetes in mice treated at early or late preclinical stages. In five-week old female mice, single dose intraperitoneal vaccinia virus expressing a cholera toxin B chain: insulin fusion protein decreases diabetes incidence from 70% to 20%. In irradiated four-week-old female mice, intravenous transfer of 1000 hematopoietic stem cells that encode proinsulin II expression in antigen-presenting cells progeny completely prevents diabetes.

39,56 57 58 59 60

Abbreviations: LCMV, lymphocytic choriomeningitis virus; Tregs, regulatory T cells; GAD, glutamic acid decarboxylase.

remission phase after diagnosis (71), and a pilot study of prophylactic systemic insulin had suggested that this approach might be of benefit in at-risk relatives (72). In DPT-1, low dose systemic insulin (daily subcutaneous injection and annual fourday intravenous infusion) was given to a high-risk group of islet antibody-positive relatives (>50% risk of diabetes over five years), matched with an untreated but closely monitored control group, but there was no effect of treatment on diabetes incidence

(67). In the subsequent DPT-1 of oral insulin, islet autoantibodypositive relatives with a 25% to 50% five-year risk of developing diabetes were randomized to receive 7.5 mg oral insulin or placebo daily for a year (69). The primary analysis revealed no effect on diabetes incidence, but a post hoc analysis revealed heterogeneity of effect according to the level of insulin autoantibodies (IAA). The subgroup with confirmed IAA more than 80 nU/mL, although progressing to diabetes at a faster rate,

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Table 5 Vaccination Trialsa for Primary Prevention of Type 1 Diabetes in Children with High Genetic Risk Primary prevention Trial design/subjects (n) Oral and nasal insulin (Prepoint study)

Follow-up (mo)

Chief investigator/sponsor Ezio Bonifacio, Center for Regenerative Therapies Dresden, Dresden University of Technology, Dresden, Germany; Juvenile Diabetes Research Foundation (JDRF), New York, U.S.A.

Three arm: oral insulin (2.5, 7.5, 22.5, 67.5 mg); nasal insulin (0.28, 0.88, 2.5, 7.5 mg) or placebo, for 10 days then twice weekly; n ¼ 40.

a

Trials that have been designed but not yet opened or are incomplete or not reported are shown in italics.

Table 6 Vaccination Trialsa for Secondary Prevention of T1D in Children or Young Adults with a T1D Relative and Islet Antibodies Secondary prevention Subjects (n)

Follow-up (mo)

Outcome

References

IV and SC insulin

14

84

66

Parenteral insulin (DPT-1) Intranasal insulin (Melbourne Intranasal Insulin Trial I)

339 38

44 48

Oral insulin (DPT-1)

372

52

Intranasal insulin (Diabetes Prediction and Prevention Project) Intranasal insulin (Melbourne Intranasal Insulin Trial II): 1.6 or 16 mg insulin or placebo daily 10 days, then weekly one year

168

Mean 21 (0–116)

Delay in onset of diabetes. No effect on islet antibody levels. No effect on diabetes development. Increased antibody and decreased T-cell responses to insulin. Stable first phase insulin response to IV glucose. Overall, no effect on diabetes development. Post hoc analysis ¼ benefit in IAA-positive subgroup. No effect on diabetes development.

264

60

Oral insulin (NIH Diabetes TrialNet) 7.5 mg insulin capsule daily

T1D relatives with IAA and another islet autoantibody. No fixed n.

No fixed period (‘‘maximum information trial’’).

67 68

69

70 Leonard C. Harrison, Royal Melbourne Hospital, Victoria, Australia; Diabetes Vaccine Development Centre (DVDC), Garvan Institute of Medical Research, Sydney, Australia. Jeffrey Krischer, University of Southern Florida, Florida, U.S.A.; TrialNet investigators

a Trials that have been designed but not yet opened, or are incomplete or not reported are shown in italics. Abbreviations: T1D, type 1 diabetes; IV, intravenous; SC, subcutaneous; DPT, Diabetes Prevention Trial; IAA, insulin autoantibodies.

showed a potential beneficial effect of oral insulin, which delayed diabetes onset by more than four years (69). Because this finding was made post hoc, it can only be hypothesis generating, and must be confirmed by a further trial, recently initiated by NIH TrialNet. Unfortunately, the opportunity has not been taken to test doses of oral insulin other than the 7.5 mg daily dose used in DPT-1. Some would contend that there is no justification for undertaking major, expensive trials without knowing that the dose of autoantigen used is at least bioactive (as judged by surrogate biomarkers), as well as safe. Coupled with dose-ranging studies is the urgent need to develop and standardize relevant biomarkers, particularly measures of pathogenic and regulatory T cells. The Melbourne intranasal insulin trial I (INIT I) (68) was a randomized crossover trial of intranasal insulin vaccine in young islet autoantibody-positive at-risk relatives (median age 10.8 years; n ¼ 38). Two 400 mg doses of insulin per nostril were self-administered daily for 10 days, then on two consecutive days each weekend, for six months. The aim was to determine if

intranasal insulin was safe and would induce changes in surrogate immune and metabolic markers consistent with an immunoprotective effect. No local or systemic adverse effects were observed. Diabetes developed in 12 subjects who had negligible b-cell function at entry, after a median of 1.1 years. b-cell function in the remaining 26, the majority of whom had antibodies to two or three islet autoantigens and first phase insulin release (FPIR) more than first percentile at entry, remained stable overall for a median follow-up of three years. Intranasal insulin was associated with an increase in insulin antibody concentrations and a decrease in T-cell proliferative responses to (denatured) insulin. Thus, this trial identified a dose of intranasal insulin that was safe and induced changes in immunity to insulin, as previously seen in NOD mice. Because it was a crossover trial in which all subjects received treatment with intranasal insulin for six months, it could not determine if intranasal insulin prevented loss of b-cell function and diabetes. This was addressed in the Diabetes Prediction and Prevention Project (DIPP) in Finland (70) and is being addressed in the

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INIT II in Australia and New Zealand, with clinical diabetes as the primary outcome measure. In DIPP, 224 infants and 40 siblings positive for two or more islet autoantibodies were randomized to receive intranasal human insulin (1 unit/kg/day) or placebo for a median of 1.8 years. The trial was terminated because there was no difference in survival between the groups. Notwithstanding this negative outcome, INIT II is continuing because there are significant differences between the two trials. First, in DIPP, the intranasal insulin dose was less than 20 units per child, lower than 40 units in the lowest dose arm of INIT II. Second, in DIPP, uncertainty about compliance was acknowledged. Third, there was a substantial withdrawal of participants after randomization in DIPP, amounting to 56 of 224 index infants (25%) and 14 of 40 siblings (35%). Next, the index infants in DIPP who developed islet autoantibodies before three years of age represent the highest risk group for diabetes. Finally, and probably related to the fact that young islet autoantibody-positive children have the most rapid rate of progression to clinical diabetes, approximately half the index cases randomized in DIPP appear to have had low FPIR to intravenous glucose, and were therefore likely to be in a late, subclinical phase when autoantigen-specific vaccination is not likely to be effective. INIT II is a randomized, placebo-controlled, three-arm trial of two doses of intranasal insulin (40 and 440 IU) administered daily for seven days, and then weekly for a year, with a further fouryear follow-up, in 264 T1D relatives aged 4 to 30 with autoantibodies to at least two islet antigens and FPIR more than 10th percentile (approximate 40% risk of diabetes over five years). In addition to the primary outcome of diabetes, secondary outcome measures are metabolic and immune markers, but results will probably not be available before 2012. In contrast to (pro)insulin, GAD65 is widely distributed, including in the brain where it is the target of autoimmunity in the rare disorder, Stiff-Person syndrome. Although this distribution would appear to potentially increase the risk of side effects, a phase II dose-finding study of subcutaneous recombinant human GAD65 (4, 20, 100, or 500 mg at weeks 1 and 4) in alum in adults with T1D (73) found no serious adverse effects. Potential metabolic benefit was seen only with the 20 mg dose. In the follow-up phase II trial (55), 70 T1D patients aged 10 to 18 years, within 18 months of diagnosis, with GAD65 autoantibodies and residual insulin secretion (fasting serum C-peptide >0.1 nM) were randomized to 20 mg of subcutaneous GAD65alum or alum alone on days 1 and 30. GAD65-alum induced GAD65-specific immune responses. C-peptide secretion to a mixed meal was similar between the groups out to 15 months (the primary endpoint), but over 30 months the decline in fasting C-peptide (0.21 vs. 0.27 nM, P ¼ 0.045) and mealstimulated C-peptide area under the curve (0.72 vs. 1.02 nM, P ¼ 0.04) were marginally less in the GAD65-alum group. No effect was seen in patients treated more than six months after diagnosis. The authors concluded, ‘‘GAD-alum may contribute to the preservation of residual insulin secretion.’’ Notwithstanding, the treatment did not alter either insulin requirement or glycemic control.

RHEUMATOID ARTHRITIS In rheumatoid arthritis (RA), the cytokines TNF-a and IL-1 mediate synovial inflammation and tissue damage and are the targets of effective passive immunotherapy with monoclonal antibodies or IL-1 receptor antagonist (IL-1Ra). A cheaper and

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more convenient alternative to repeated passive antibody treatment is autovaccination to generate blocking antibodies, with the proviso of safety. Vaccination of mice with either TNF-a or IL-1a or b chemically cross-linked to virus-like particles of the bacteriophage Qb induced a rapid and long-lasting autoantibody response (74,75). The IL-1b conjugate protected collageninduced arthritis mice with greater efficacy than daily highdose IL-1Ra (75). A similar outcome was reported after vaccination with plasmid encoding TNF-a (76) or macrophage migration inhibitory factor (MIF) (77). These findings are encouraging for trials in RA and in other ‘‘factor’’-mediated diseases, but it is critical to ensure that such vaccines do not impair innate immune protection against infection and cancer. Trials of oral vaccination in RA have been reviewed (78). RCTs of oral bovine or chicken type II collagen (CII) in RA initially suggested benefit but have not been validated. In 60 patients with severe, active disease, a decrease in the number of swollen and tender joints occurred in those fed CII for three months but not in those fed placebo. Four patients in the CII group had complete remission of the disease. No side effects were evident (79). In 280 RA patients given chicken CII 20 to 2500 mg daily for six months, significant benefit was reported at the lowest dose (80). Subsequently, these investigators, through AutoImmune Inc., carried out a 760 patient phase III trial of oral CII (‘‘Colloral’’) 60 mg daily, in which there was a large placebo effect and no difference between groups. On the other side of the Atlantic, 55 patients with active RA who had failed treatment with at least one slow-acting drug were fed 0.05 mg, 0.5 mg, or 5 mg of bovine CII or placebo daily for six months (81). Drugs were stopped, although prednisolone was permitted at less than 10 mg/day. Only the 0.5 mg/day dose had a small but significant disease improvement effect. Thus, the clinical efficacy of oral CII in established RA is uncertain. Further trials of oral CII, or other RA autoantigens, are warranted in very early RA or in those at risk.

MULTIPLE SCLEROSIS The myelin antigens myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) have been used both to induce and suppress the development of EAE in rodents (82,83), being the basis for vaccination trials in humans with multiple sclerosis (MS). The autovaccination strategy to block disease mediators as described in arthritis models has also been applied in mouse models of MS. Vaccination with an IL-17A-OVA conjugate elicited long-lasting, selective inhibition of IL-17A activity in vivo, which completely prevented EAE induced by PLP peptide 139 to 151 in SJL mice (84). The same investigators sounded an important note of caution, however, from similar studies in which the target was IL-12 p40 subunit. Although protective against EAE, vaccination against IL-12 p40 resulted in mice being unable to control infection with Leishmania major (85). Oral bovine myelin was reported to induce TGF-bsecreting T cells to MBP and PLP in humans (86). In an RCT conducted by Autoimmune Inc, a single 300 mg dose of bovine myelin had no effect on relapsing-remitting MS. By subcutaneous injection, an analogue polypeptide of MBP called glatiramer acetate or copolymer I (Copaxone), possibly acting as an altered peptide ligand (87), has not lived up to expectations in a Cochrane analysis of RCTs (88). In a phase I/IIa open label study by Apitope Technologies Ltd. (Bristol, U.K.) (89), a mixture of four soluble peptides from MBP (ATX-MS-1467)

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was injected in dose-escalation up to 800 mg in six progressive MS patients, without adverse effect. This treatment was said to induce IL-10þ Tregs and specifically to reduce the T-cell response to MBP. A phase II RCT in relapsing-remitting MS is planned. In a phase II trial by Bayhill Therapeutics (California, U.S.) (90), plasmid-based DNA encoding MBP was injected intramuscularly in a dose of either 0.5 or 1.5 mg four-weekly over 44 weeks and compared with saline placebo in 267 patients with relapsing-remitting MS (91). At the 0.5 mg dose, the occurrence rate of new gadolinium-enhancing lesions on brain magnetic resonance imaging at 8 to 48 weeks was 61% lower (p ¼ 0.05), and the mean volume of enhancing lesions at 48 weeks was 51% lower (p ¼ 0.02), compared with placebo. These changes were accompanied by reductions in myelinspecific autoantibodies. Intriguingly, no effects were seen at the 1.5 mg dose. This narrow therapeutic window is reminiscent of that with subcutaneous GAD65 in T1D (73) and oral CII in RA (81). They could be examples of ‘‘low-zone tolerance’’ described in the early days of modern immunology (92,93), in which case careful dose-ranging studies will be required to optimize autoantigen-specific immunotherapy. Vaccination with autoantigen- or disease-specific T cells or TCR peptides is a ‘‘personalized’’ strategy that has been tested with varying success in several rodent models of autoimmune disease and translated in particular to human MS. Myelin antigen-reactive T cells are selected and expanded from individual MS donors, irradiated, and then reinjected, resulting in deletion or regulation of the targeted T cells in vivo (94). Vaccination with TCR peptides appeared to enhance peptidespecific natural CD4þ CD25þ Tregs in MS subjects (95). Small trials have reported reduced relapse rates over relatively short follow-up periods, without adverse effects, but validation is awaited from several larger controlled trials now in progress (96).

CRITIQUE OF AUTOANTIGEN-SPECIFIC VACCINATION TRIALS IN HUMANS Why have trials of autoantigen-specific vaccination, mostly by oral administration in established T1D, MS, and RA, shown no clinical effect? There are several possible reasons. First, on the basis of experience in the rodent models, this approach will probably not work alone in end-stage clinical disease. Further investigation is required to determine if it would be effective as ‘‘combinatorial’’ therapy with agents that inactivate or delete pathogenic effector cells. If a balance between protective and pathogenic T cells determines clinical outcome, then autoantigen-specific vaccination should be most effective before the onset of disease. Second, in the case of T1D at least, the dose of oral insulin, 7.5 mg a day, was probably inadequate. In prediabetic NOD mice, milligrams of gavaged insulin were required to induce regulatory CD4þ T cells and partially suppress diabetes development. On a body weight basis, the 7.5 mg dose given to humans in these trials (and also to at-risk individuals in DPT-1) equates to only a few micrograms in the mouse. Third, also related to dose, the route of administration may be important. Proteins are degraded in the stomach, limiting bioavailability to the upper small intestine. On the other hand, the nasal route delivers protein directly to the mucosa, and experimental evidence shows that nasal administration may be more efficient in eliciting a mucosal immune response (41,97). A further reason why human trials could have failed to reveal clinical effects is that antigen is a ‘‘double-edged

sword.’’ We observed that nasal, aerosol, and oral OVA all induced not only mucosal tolerance but also pathogenic CD8þ T cells capable of destroying b cells expressing transgenic OVA (41). Therefore, to optimize protection, autoantigen protein or peptide should be selected or modified to avoid potentially pathogenic MHC class I-restricted epitopes. A major deficiency of all human trials is the absence of markers of bioavailability and efficacy as secondary endpoints, in addition to clinical outcomes. It is not surprising that endstage disease is resistant to vaccination therapy; nevertheless, potential efficacy could be gauged by showing that autoantigen administration has a bioeffect. In INIT I (68), ex vivo antibody and T-cell responses to insulin demonstrated that intranasal insulin was bioavailable, and the immune changes were consistent with mucosa-mediated tolerance. More recently, in INIT III, an RCT of intranasal insulin in adults with recent onset, non-insulin requiring T1D, we observed more striking evidence of tolerance induction. In participants in whom deteriorating metabolic control necessitated treatment with insulin, the insulin antibody response resulting from injected subcutaneous insulin was significantly blunted in those who had received intranasal insulin compared with placebo (Fourlanos and Harrison, unpublished data). In summary, although autoantigen-specific vaccination is a rational strategy for promoting natural immunoregulatory mechanisms, its successful translation to humans will depend on careful attention to the selection of autoantigen protein, peptide, or DNA to minimize induction of pathogenic immunity; dose-finding studies; surrogate biomarkers in addition to clinical endpoints; and application to individuals at genetic risk or in the early phase of subclinical disease.

VACCINATION FOR OTHER CHRONIC INFLAMMATORY DISORDERS Vaccination is a therapeutic option for a range of other noninfectious inflammatory disorders in which mediators of pathology can be identified as immune targets (98). Examples include allergy, obesity, insulin resistance, atherosclerosis, Alzheimer’s disease, and ‘‘stroke.’’ Although these disorders are associated with evidence for low-grade inflammation, except in the case of allergy, the etiological role of inflammation remains to be firmly established. Vaccination is, however, a way to address this question. Alzheimer’s disease is discussed in Chapter 87. Tumor immunotherapy by vaccination, a major separate topic, is discussed in Chapter 85; however, low-grade inflammation is implicated in predisposing to cancer (99).

Obesity Obesity is a serious public health problem in economically privileged and emergent societies, with more than one billion people worldwide being overweight and at risk of associated effects including T2D, hypertension, cardiovascular disease and stroke, osteoarthritis, and cancer. Education and behavior change are unlikely to be effective at a population level in the near term and current medical therapies are ineffective. Vaccination to modulate the bioavailability of peptide hormones that regulate appetite, food intake, and energy balance is a novel approach to the treatment of obesity. Two candidate hormones are ghrelin and gastric inhibitory peptide (GIP, also known as glucose-dependent insulinotropic polypeptide). Ghrelin is secreted in the periphery by cells in the stomach and acts at

Chapter 88: Vaccination for Autoimmune and Other Chronic Inflammatory Disorders

the arcuate nucleus and ventromedial hypothalamus to increase appetite (100). Vaccination of adult rats with ghrelin peptides conjugated to keyhole limpet hemocyanin-induced antibodies that bound the circulating active Ser-3-(n-octanoyl) form of ghrelin and decreased feeding efficiency, relative adiposity, and body weight gain compared with control rats (101). GIP is an incretin produced by K cells in the duodenum and upper jejunum, which stimulates insulin biosynthesis and secretion (102). Recently, GIP receptor-deficient mice were shown to be completely protected from diet-induced obesity (103), suggesting that blockade of GIP signaling could be a therapeutic strategy for the treatment of obesity. Vaccination of mice with GIP peptides covalently attached to virus-like particles induced high titers of specific antibodies and efficiently reduced body weight gain in animals fed a high-fat human diet (104). Importantly, despite the incretin action of GIP, vaccinated mice were not glucose intolerant. Further preclinical safety/toxicology studies will be required before this strategy is translated to humans.

Insulin Resistance and Type 2 Diabetes Tissue resistance to the action of insulin is a characteristic of obesity and contributes to the pathogenesis of T2D. Many investigators have shown that insulin resistance is associated with chronic, low-grade innate immune inflammation in multiple tissues (105,106) that resolves with weight loss (107,108). Metabolic stress in adipocytes associated with the accumulation of fat induces release of chemoattractants including monocyte chemoattractant protein-1 (MCP-1 or CCL2), which recruits circulating monocytes to adipose tissue and promotes their differentiation into macrophages (109). In adipose tissue of obese mice, the major source of inflammatory mediators, macrophages, are hypothesized to underlie insulin resistance (110,111). Innate immunity may also impair pancreatic b-cell function in T2D. Islets from humans with T2D contain more macrophages and secrete more proinflammatory cytokines than normal islets (112). The role of inflammation in insulin resistance and T2D is supported by trials of anti-inflammatory agents. Salicylates were documented to improve glucose tolerance over a century ago (113,114). RA patients who underwent treatment with TNF-a monoclonal antibody exhibited improved insulin sensitivity (115) and recombinant IL-1Ra was shown to improve insulin secretion and glucose tolerance in T2D (116). Antagonists of chemokine receptor 2 (CCR2), the receptor for MCP-1, including small molecules in commercial development, have shown promise in reversing fat accumulation, insulin resistance, and glucose intolerance in mice (117). Although one or more specific cytokines or chemokines have not been proven to fully account for insulin resistance, autovaccination against candidate mediators is an obvious potential therapeutic strategy.

Atherosclerosis Many studies have demonstrated a role for innate and adaptive immunity in atherosclerosis (118,119). The rationale for vaccination in atherosclerosis (120) is therefore the same as in obesity-insulin resistance diabetes; indeed, a continuum of inflammatory mechanisms leads from this ‘‘metabolic syndrome’’ to its vascular complications. Moreover, it is likely that drugs used for cardiovascular prophylaxis, including cholesterol-lowering agents, have significant anti-inflammatory effects critical for their efficacy.

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In rodent models and humans with atherosclerosis, oxidized low-density lipoproteins (oxLDL), heat shock protein (HSP) 60/65 and b-2 glycoprotein 1 have been identified as targets of humoral and cellular immunity, and protection has been afforded by vaccination with these autoantigens. Autoantibodies against malondialdehyde (MDA)-modified lysine, an epitope in oxLDL, occur naturally and are present as immune complexes with oxLDL in atherosclerotic lesions. Atherosclerosis-prone LDL receptor-deficient rabbits vaccinated with homologous MDA-LDL generated high titers of antibodies with similar specificity to the naturally occurring autoantibodies and exhibited significant reduction of aortic tree atherosclerotic lesions after 6.5 months compared with controls (121). It has been proposed that pneumococcal vaccination decreases atherosclerosis by generating IgM antibodies that cross-react with oxLDL (122), but this is disputed in humans (123). Separately, influenza infection has been associated with an increased risk of acute cardiac infarction, and influenza vaccination has been reported to reduce this risk (124), but the protective effect of vaccination has been questioned (125). Autoantibodies to apolipoprotein (apo) B-100 peptides are present in humans and have been shown to be associated with decreased cardiovascular risk. To determine if apo B-100 peptide vaccines are protective in mice expressing human apo B-100, LDL receptor-deficient/human apo B-100 transgenic mice were injected subcutaneously (SC) with native human apo B-100 peptides in alum at 6, 9, and 11 weeks of age (126). Treatment significantly reduced atherosclerosis independent of preexisting apo B-100 peptide autoantibodies and without an increase in peptide-specific IgG, suggesting that it was mediated by cellular immune responses. Following the discovery of immunity to HSP 60/65 (127), nasal administration of mycobacterial HSP 65 to LDL receptordeficient mice maintained on a high-cholesterol diet was shown to be associated with a decrease in atherosclerotic plaque size and macrophage and T-cell numbers in the aortic arch, with an increase in colocalized interleukin-10 expression. A similar trend was observed in orally treated mice (128). Inhibition of cholesterol ester transfer protein (CETP) prevents the transfer of cholesterol ester from high-density lipoprotein (HDL) to triglyceride-rich lipoproteins in exchange for triglyceride, thereby raising the level of HDL, which is protective against atherosclerosis. In a phase I human trial (129), 8 of 15 subjects (53%) who received two injections of CETP vaccine (which reduced atherosclerosis in rabbits) developed anti-CETP antibodies. Short-term adverse effects were absent, but no follow-up studies have been reported. Recently, intranasal plasmid encoding CETP coupled to chitosan nanoparticles was shown to induce anti-CETP antibodies and significantly reduce atherosclerosis in cholesterol-fed rabbits (130). These cited examples of proof-of-concept for vaccinationinduced protection against atherosclerosis in animal models are a strong foundation for human trials. The landscape of emerging vaccines for human atherosclerosis has recently been surveyed (131).

EPILOGUE The close of the last millennium was the golden age of vaccination for prevention of infectious diseases, despite remaining challenges in specific cases such as malaria and HIV. As we advance into the new millennium, vaccination both in concept and practice is no longer bounded by notions of

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nonself versus self, and is seen as a therapeutic strategy for a host of noninfectious diseases, in particular those involving chronic inflammation. There is one important caveat. Vaccination is highly specific, relatively inexpensive, and generally safe when the antigen is nonself; however, when the antigen is self, it is a potential double-edged sword. Vaccination holds enormous promise for emergent global epidemics of noninfectious diseases such as diabetes and atherosclerosis, but meeting this promise with minimum harm will require the continuing pursuit of basic knowledge and the development of improved immunological tools to guide clinical trials.

ACKNOWLEDGMENTS I thank my students and colleagues, past and present, for their inspiration and support. Catherine McLean provided administrative and secretarial assistance. My research is funded by Program Grants from the National Health and Medical Research Council of Australia (NHMRC) (516700) and the Juvenile Diabetes Research Foundation International (JDRF) (7-2006-318), and I am a Senior Principal Research Fellow of the NHMRC.

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100. Tscho¨p M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407(6806):908–913. 101. Zorrilla EP, Iwasaki S, Moss JA, et al. Vaccination against weight gain. Proc Natl Acad Sci U S A 2006; 103:13226–13231. 102. Ballinger A. Gastric inhibitory polypeptide links overnutrition to obesity. Gut 2003; 52:319–320. 103. Miyawaki K, Yamada Y, Ban N, et al. Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med 2002; 8:738–742. 104. Fulurija A, Lutz TA, Sladko K, et al. Vaccination against GIP for the treatment of obesity. PLoS ONE 2008; 3:e3163. 105. Pickup JC. Inflammation and activated innate immunity in the pathogenesis of type 2 diabetes. Diabetes Care 2004; 27:813–823. 106. Hotamisligil GS. Inflammation and metabolic disorders. Nature 2006; 444:860–867. 107. Bruun JM, Helge JW, Richelsen B, et al. Diet and exercise reduce low-grade inflammation and macrophage infiltration in adipose tissue but not in skeletal muscle in severely obese subjects. Am J Physiol Endocrinol Metab 2006; 290:E961–E967. 108. Cancello R, Henegar C, Viguerie N, et al. Reduction of macrophage infiltration and chemoattractant gene expression changes in white adipose tissue of morbidly obese subjects after surgeryinduced weight loss. Diabetes 2005; 54(8):2277–2286. 109. Weisberg SP, Hunter D, Huber R, et al. CCR2 modulates inflammatory and metabolic effects of high-fat feeding. J Clin Invest 2006; 116(1):115–124. 110. Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, et al. Macrophage-specific PPARgamma controls alternative activation and improves insulin resistance. Nature 2007; 447(7148):1116–1120. 111. Patsouris D, Li PP, Thapar D, et al. Ablation of CD11c-positive cells normalizes insulin sensitivity in obese insulin resistant animals. Cell Metab 2008; 8(4):301–309. 112. Ehses JA, Perren A, Eppler E, et al. Increased number of isletassociated macrophages in type 2 diabetes. Diabetes 2007; 56(9): 2356–2370. 113. Ebstein W. Zur therapie des diabetes mellitus, insbesondere uber die anwendeng der salicylauren natron bei demselben. Berl Klin Wochenschr 1876; 13:337–340. 114. Williamson R. On the treatment of glycosuria and diabetes mellitus with sodium salicylate. Br Med J 1901; 1:760–762. 115. Kiortsis DN, Mavridis AK, Vasakos S, et al. Effects of infliximab treatment on insulin resistance in patients with rheumatoid arthritis and ankylosing spondylitis. Ann Rheum Dis 2005; 64:765–766. 116. Larsen CM, Faulenbach M, Vaag A, et al. Interleukin-1-receptor antagonist in type 2 diabetes mellitus. N Engl J Med 2007; 356 (15):1517–1526. 117. Tamura Y, Sugimoto M, Murayama T, et al. Inhibition of CCR2 ameliorates insulin resistance and hepatic steatosis in db/db mice. Arterioscler Thromb Vasc Biol 2008; 28(12):2195–2201. 118. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340:115–126. 119. Hansson GK. Immune mechanisms in atherosclerosis. Arterioscler Thromb Vasc Biol 2001; 21:1876–1889. 120. Shah PK, Chyu KY, Fredrikson GN, et al. Vaccination for atherosclerosis: a novel therapeutic paradigm. Expert Rev Vaccines 2004; 3:711–716. 121. Palinski W, Miller E, Witztum JL. Immunization of low-density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad Sci U S A 1995; 92:821–825. 122. Binder CJ, Ho¨rkko¨ S, Dewan A, et al. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med 2003; 9:736–743. 123. Damoiseaux J, Rijkers G, Tervaert JW. Pneumococcal vaccination does not increase circulating levels of IgM antibodies to oxidized LDL in humans and therefore precludes an anti-atherogenic effect. Atherosclerosis 2007; 190:10–11.

Chapter 88: Vaccination for Autoimmune and Other Chronic Inflammatory Disorders 124. Madjid M, Awan I, Ali M, et al. Influenza and atherosclerosis: vaccination for cardiovascular disease prevention. Expert Opin Biol Ther 2005; 5:91–96. 125. Jackson LA, Yu O, Heckbert SR, et al. Vaccine Safety Datalink Study Group. Influenza vaccination is not associated with a reduction in the risk of recurrent coronary events. Am J Epidemiol 2002; 156:634–640. 126. Fredrikson GN, Bjo¨rkbacka H, So¨derberg I, et al. Treatment with apo B peptide vaccines inhibits atherosclerosis in human apo B100 transgenic mice without inducing an increase in peptidespecific antibodies. J Intern Med 2008; 264:563–570. 127. Wick G, Schett G, Amberger A, et al. Is atherosclerosis an immunologically mediated disease? Immunol Today 1995; 16:27–33.

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128. Maron R, Sukhova G, Faria AM, et al. Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density lipoprotein receptor-deficient mice. Circulation 2002; 106(13):1708–1715. 129. Davidson MH, Maki K, Umporowicz D, et al. The safety and immunogenicity of a CETP vaccine in healthy adults. Atherosclerosis 2003; 169:113–120. 130. Yuan X, Yang X, Cai D, et al. Intranasal immunization with chitosan/pCETP nanoparticles inhibits atherosclerosis in a rabbit model of atherosclerosis. Vaccine 2008; 26:3727–3734. 131. Riley E, Dasari V, Frishman WH, et al. Vaccines in development to prevent and treat atherosclerotic disease. Cardiol Rev 2008; 16:288–300.

89 Immunotherapies To Treat Drug Addiction Paul R. Pentel Department of Medicine, Hennepin County Medical Center, University of Minnesota Medical School, Minneapolis, Minnesota, U.S.A.

Dan E. Keyler Department of Medicine, Hennepin County Medical Center and College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, U.S.A.

Thomas R. Kosten Department of Psychiatry and Neuroscience, Baylor College of Medicine, Michael DeBakey Veterans Affairs Medical Center, Houston, Texas, U.S.A.

RATIONALE

MECHANISM OF ACTION

Drugs of abuse produce their addictive effects by acting on neural pathways in the brain. Currently available medications for drug addiction act by targeting these pathways and altering their response to the addictive drug. The challenge posed by this treatment strategy is that the neural pathways that are important in drug abuse are also important in mediating a myriad of other natural functions ranging from cognition to emotion. In altering these pathways to treat drug abuse, medications may alter these natural functions, leading to side effects and limitations on the dose of medication that can be administered. Immunotherapies (vaccination or passive immunization) offer an alternative strategy for the treatment of drug abuse in which the drug itself is the target rather than the brain. Vaccines directed against these drugs elicit the production of drugspecific antibodies, which bind and sequester drug, and reduce its distribution to brain. By virtue of acting outside the brain, vaccines appear to circumvent the central nervous system side effects that limit the usefulness of other therapies. The specificity of vaccination in binding only the drug of interest, and the generally excellent safety profile of vaccination suggest that side effects outside of the central nervous system should also be minimal. Practical features of vaccination such as an anticipated long-lasting effect and avoiding the need for daily medication could also prove helpful. The concept of immunotherapy to treat drug abuse was introduced over 30 years ago. A vaccine against heroin was studied in monkeys (1,2) but further investigation was not pursued because of the development of other promising therapies for heroin addiction at the time. Interest in this area was rekindled in the 1990s with reports that cocaine vaccines could block a variety of cocaine effects in rats (3–6). Vaccines are currently being investigated for the treatment of cocaine, nicotine, phencyclidine (PCP), methamphetamine, and heroin abuse. A cocaine vaccine and three nicotine vaccines have reached phase I or II clinical trials, with preliminary indications of efficacy. This chapter will consider the mechanistic basis for using vaccines to treat drug addiction and their application to specific drugs.

The vaccines discussed below act by producing antibodies that bind the addictive drug and alter its tissue distribution or elimination. Both active immunization of the animals being studied (herein referred to as vaccination) and passive immunization (administration of exogenously produced antibodies) have been studied. An alternative approach involving the generation of catalytic antibodies that act by enhancing drug metabolism has also been applied to cocaine.

Relationship Between Addiction and Drug Delivery to Brain Two key features of addictive drugs are that they are rewarding (produce pleasant subjective effects) and reinforcing (their use leads to further drug seeking and self-administration). In addition, abstinence after prolonged drug use can result in a withdrawal syndrome. Each of these features of addiction is influenced by the pharmacokinetics of the drug: its dose, rate of delivery to the brain, and rate of disappearance from the brain. Within limits, higher doses and faster delivery to the brain are associated with increased reward and reinforcement. Faster drug elimination is associated with a greater severity of withdrawal and faster return of drug craving after a dose. Immunotherapies can affect each of these processes. When an addictive drug is administered, it enters blood and distributes to tissues throughout the body, including the brain (Fig. 1). In an immunized animal, drug-specific antibody is present in blood and extracellular fluid. Antibody is largely excluded from the brain by the blood-brain barrier owing to its large size. Drug-specific antibody presents a potential reservoir for binding drug outside the central nervous system and before it enters the brain. When an immunized animal receives the drug in question, it is bound to antibody and sequestered in blood and extracellular fluid. If a sufficient binding capacity is available, the concentration of unbound drug is reduced. Since only unbound drug can enter the brain, the concentration of drug reaching brain is correspondingly reduced.

Chapter 89: Immunotherapies To Treat Drug Addiction

Figure 1 Effects of vaccination on drug distribution, illustrated for nicotine. (Top panel) When a smoker or experimental animal is exposed to nicotine, the drug distributes first to blood and subsequently to tissues, including the brain. (Middle panel) Vaccination elicits nicotine-specific antibodies which are located largely in blood and extracellular fluid, and which are excluded from the brain by the blood-brain barrier owing to their size. (Bottom panel) In a vaccinated animal, nicotine binds to nicotine-specific antibodies in blood (and extracellular fluid), the unbound nicotine concentration is reduced and drug distribution to brain is reduced or slowed.

Altered drug distribution is illustrated in Figure 2, in which vaccinated rats received a single nicotine dose equivalent on a weight basis to the nicotine absorbed from two cigarettes by a smoker (7). The blood nicotine concentration was increased in vaccinated animals, reflecting the binding and

Figure 2 Vaccination reduces nicotine distribution to brain. Rats were vaccinated with nicotine immunogen or control immunogen over a period of seven weeks. One week after the vaccination schedule was complete, nicotine 0.03 mg/kg was administered intravenously. This dose is equivalent on a weight basis to the nicotine absorbed by a smoker from two cigarettes. Serum and brain were sampled three minutes after the injection. The serum nicotine concentration was markedly increased in vaccinated rats due to the binding of nicotine to antibody in serum. Brain nicotine concentrations were reduced by 60%. Source: From Ref. 7.

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Figure 3 Effects of serum antibody titers on nicotine distribution to brain (protocol as in Figure 2). Brain nicotine concentrations in vaccinated rats were lowest in animals with the highest serum antibody titers (a measure of antibody concentrations). Source: From Ref. 8.

sequestration of nicotine in blood. Brain nicotine concentration was decreased by two-thirds compared to nonvaccinated controls. Brain nicotine concentration was lowest in those animals with the highest nicotine-specific serum antibody titers (Fig. 3), illustrating the critical importance of reliably producing high serum antibody concentrations to optimize the efficacy of this treatment strategy (8). The increased concentration of nicotine in blood in vaccinated rats does not result in increased peripheral effects of nicotine because the antibody-bound drug is pharmacologically inactive (7). In the study illustrated in Figure 2, the estimated total body nicotine-specific antibody content of vaccinated rats was approximately equal to the moles of nicotine administered. Smokers take in considerably greater nicotine doses throughout the day, (9) raising the question of whether the antibodies will become saturated and therefore ineffective in a regular smoker. Surprisingly, the ability of vaccination to reduce drug distribution to brain persists even when single nicotine doses exceed the estimated binding capacity of antibody by up to 67-fold (10), or in the presence of continuous infusion of nicotine at rates equivalent to two to three packs of cigarettes daily (11). A similar unexpected efficacy in the face of very large drug doses has also been observed for cocaine (12) and PCP (13) antibodies, and appears to be a general feature of immunotherapy for drugs of abuse. The basis for this unanticipated pharmacokinetic efficacy is not well understood but is clearly important for the successful clinical use of vaccination, because both the single and daily doses of most addictive drugs equal or exceed the calculated binding capacity that can be provided by vaccination (Table 1). One contributing factor is that vaccination or passive immunization reduces nicotine distribution to brain to a greater extent than to other organs, and this has been shown for PCP as well (13). A second possible contributor to the unexpected efficacy of immunization despite high drug doses is that immunization may also slow drug distribution to brain. When rats are given repeated bolus doses of nicotine over days to weeks to simulate regular cigarette smoking, the chronic accumulation of nicotine in brain is not altered by immunization; brain nicotine

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Table 1 Typical Drug Doses Generally Exceed the Anticipated Binding Capacity of Drug-Specific Antibody in a Vaccinated Human

Drug dose Nicotine Single dose 0.015 mg/kg (9.3
108 mol/kg) Daily dose 0.3 mg/kg (1.9
106 mol/kg) Phencyclidine Single dose 0.14 mg/kg (5.6
107 mol/kg) Daily dose 0.7 mg/kg (2.8
106 mol/kg) Cocaine Single dose 0.5 mg/kg (1.6
106 mol/kg) Daily dose 5 mg/kg (1.6
105 mol/kg) Methamphetamine Single dose 0.1 mg/kg (5
107 mol/kg) Daily dose 10 mg/kg (5
105 mol/kg)

Ratio of drug to estimated binding capacity 0.3 7 2.1 10 6 59 1.9 190

The calculated binding capacity of 2.7
107 mol/kg is extrapolated from animal studies and assumes that 1% of total antibody (IgG) is specific for the drug in question and that there are two drug-binding sites per molecule of IgG. Source: From Ref. 7.

levels are the same as in nonvaccinated rats. However, the early peak level produced by each individual nicotine dose (3–25 minutes after the dose) is reduced by up to 50%. Thus nicotine entry into brain in the setting of chronic dosing is slowed rather than prevented. Since immunization is effective in blocking nicotine self-administration in rats (see below), the slowing of distribution to brain may be sufficient to have a substantial behavioral impact. Whether immunotherapies for other addictive drugs also slow drug distribution to brain is not yet clear. The effects of vaccination on drug elimination differ among drugs. The binding of nicotine by antibody makes it less available for metabolism and slows its elimination half-life in rats by three- to six-fold (14). This could be a detrimental effect in that slower elimination would favor accumulation of nicotine and saturation of antibody. On the other hand, genetically determined slower nicotine elimination in humans is associated with smoking fewer cigarettes per day and longer intervals between cigarettes, perhaps because the effects of each cigarette last longer (15). It is possible that similar effects contribute to the ability of immunotherapies to block the behavioral effects of nicotine. In contrast, cocaine’s elimination half-life does not appear to be altered by vaccination (12).

VACCINE FORMULATION AND VACCINATION SCHEDULES Overview There are three principal requirements of vaccines used to treat drug addiction. First, the antibodies elicited by vaccination must have a high enough affinity to effectively bind the drug. Second, because high ratios of antibody to drug are most effective, a vaccine must elicit and maintain high concentrations of antibody throughout the period of clinical interest. Third, antibodies must be highly specific for the drug of interest. Crossreactivity with endogenous compounds could lead to toxicity, and cross-reactivity with endogenous or exogenous compounds or drug metabolites could reduce the available binding capacity for the targeted drug. However, in some cases binding of a metabolite may be desired; methamphetamine is metabolized

in part to amphetamine, which is also addictive, and an immunogen eliciting antibodies which bind both parent drug and metabolite has been developed.

Linker Drugs of abuse (molecular weights 150–300) are too small to elicit an immune response but can be rendered immunogenic by linking either the drug itself or a structurally related compound (hapten) to an immunogenic carrier protein to form a complete immunogen. After initial (primary) vaccination, periodic booster doses with the complete immunogen are needed to maintain satisfactory antibody titers: exposure to the drug by itself does not elicit an anamnestic or booster response (16,17). The conjugation or linking of drug to carrier protein has generally been accomplished using five to six atom spacers of various compositions (6,7,18). While some linkers anecdotally seem to produce better immunogens than others, no structural rule has emerged to predict which linkers are most effective. Few published data are available comparing linkers, linker strategies, or haptenation ratios (molar ratio of drug: carrier protein). A haptenation ratio of 20:1 was used for conjugating PCP to bovine serum albumin (19).

Carrier Protein A variety of carrier proteins have been used for these vaccines including bovine albumin (6,19), keyhole limpet hemocyanin (8), cholera toxin subunit (17), recombinant Pseudomonas aeruginosa exoprotein A (7), and a virus-like particle (20). Consistent superiority of any one of these over the others has not been demonstrated.

Vaccination Schedules Vaccination schedules in rats have generally involved two to four doses of vaccine over a period of 4 to 10 weeks. Clinical trials of nicotine and cocaine vaccines have used a variety of similar schedules, with booster doses three to six months later. Maximal antibody titers were reached 1 week after the third injection, decreased by about 50% two months later, and were nearly back to baseline 6 to 10 months after the final injection (16,17). This was true even for subjects who continued to smoke or use cocaine, confirming that the drug itself is not immunogenic and that booster doses will be needed to maintain antibody levels over longer durations. Because of the several-month delay in achieving high antibody concentration in serum, it may be desirable to vaccinate individuals before they stop their drug use. Both rat data (11,21,22) and clinical trials (16,17) have shown that concurrent drug use does not interfere with vaccine immunogenicity, making this approach feasible.

Antibody Concentrations in Serum In rats, a cocaine-specific serum antibody concentration of 0.05 mg/mL represents a threshold for efficacy as measured by suppression of cocaine self-administration (22,23). Thresholds for other drugs are not known, but serum drug-specific antibody concentrations of 0.1 to 0.2 mg/mL were effective in altering nicotine pharmacokinetics and effects in rats, and the magnitude of effect was related to antibody titer. Antibody titers in humans given vaccines for infectious diseases are generally lower than those achieved in experimental animals, perhaps in part because only alum adjuvant and the

Chapter 89: Immunotherapies To Treat Drug Addiction

intramuscular route have been used in humans (rather than Freund’s adjuvant administered intraperitoneally or by other routes in animals). In clinical trials of nicotine and cocaine vaccines, serum antibody concentrations have been highly variable but the highest have been approximately 0.1 mg/mL. As in rats, greater clinical efficacy appears to be strongly correlated with higher antibody concentrations (see below). This highlights the importance of developing methods to achieve high antibody titers to maximize the impact of this treatment strategy.

PASSIVE IMMUNIZATION Monoclonal antibodies or immune serum have been used to study passive immunization for addiction. Effects of passive immunization are generally similar to those of vaccination, except that the effect is immediate since stimulation of antibody production is obviated, and the dose of antibody can be controlled. As serum antibody concentrations achievable with vaccination are limited to 1% to 2% of total IgG, passive immunization can produce greater effects than vaccination if a suitably large antibody dose is used. For example, a nicotine monoclonal antibody at doses of 80 or 240 mg/kg reduced nicotine distribution to brain to a greater extent than had been previously observed with vaccination (24). An additional potential advantage of passive immunization is that antibodies with desired specificities, such as cross-reactivity with an active drug metabolite, can be selected. Doses of PCP or methamphetamine monoclonal antibodies of up to 1 g/kg have been well tolerated in rats, and could be humanized for clinical use (25).

SPECIFIC DRUGS Nicotine Overview Cigarette smoking kills 400,000 people each year in the United States (1 out of 5 deaths) and 10 times that many worldwide (26). New medications and counseling have helped many smokers quit, but the majority of those who try are still unsuccessful (27). Addiction to nicotine is the primary reason why people smoke (26). An intervention that could reduce the reinforcing effects of nicotine and render it less addictive is therefore of interest (28). Nicotine distribution to brain is substantially reduced by vaccination after clinically relevant single doses of nicotine alone or against a background of chronic nicotine dosing. Magnitude of effect is related to antibody titer or concentration in blood (7,8,11). In assessing the potential clinical usefulness of vaccination to treat nicotine addiction, key questions are (i) does vaccination reduce the reinforcing effects of nicotine? (ii) can beneficial effects be attained with antibody titers that are achievable in humans? and (iii) will vaccinated animals or humans try to compensate for reduced nicotine distribution to brain by simply taking in more nicotine, thereby overcoming the effects of vaccination? Additionally, it is possible that vaccination might affect various aspects of nicotine addiction differentially. Smokers who quit and then relapse to smoking typically do so by starting with a few puffs or a few cigarettes, a relatively small nicotine dose. Vaccination could block the reinforcing effects of those first few puffs and make them less enjoyable, thus making relapse less likely. By contrast, using vaccination to help initiate smoking cessation would involve the larger nicotine dose associated with regular daily smoking. Because the ratio of antibody

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to drug is important in determining vaccine efficacy, vaccination might be more effective for relapse prevention than for initiating smoking cessation. Active Versus Passive Immunization Vaccination of smokers is attractive as a potential clinical intervention because it is less expensive and longer lasting than passive immunization. Passive immunization has been used experimentally primarily as a methodological expedient, a means of controlling antibody dose or producing immediate effects without having to wait the one to two months required for vaccination. However, rapid onset of effect and control of dose could also be useful in treating patients, and a clinical role for passive immunization alone or as an adjunct to vaccination is possible (28). Vaccines At least eight nicotine vaccines have been studied (7,14,20, 29–33). Although not all are well described, they generally consist of nicotine conjugated to a carrier protein or virus-like particle through a short linker. Placing the linker on the nicotine distant from the major sites of nicotine metabolism allows the production of antibodies that show high specificity for nicotine (<3% cross-reactivity with the major nicotine metabolites cotinine and nicotine-N-oxide). Cross-reactivity for one nicotine vaccine with acetylcholine, the endogenous ligand for nicotinic receptors, was negligible (7). The Kd of elicited antibodies for nicotine has ranged from 20 to 40 nM (7,11). Typical serum nicotine concentrations in smokers are higher than these Kds (50–500 nM), suggesting that the saturation of nicotine-specific antibodies in smokers should be high and most of the binding capacity will be utilized. Efficacy Passive immunization of rats with nicotine-specific IgG blocked the ability of nicotine to increase blood pressure in a doserelated manner (7). Because this effect is largely mediated by peripheral autonomic ganglia, these data confirm that nicotine bound to antibody in serum is inactive. Both active and passive immunization of rats attenuates a variety of nicotine-induced behaviors that are relevant to addiction. These include the release of dopamine in the nucleus accumbens of the brain (a key mediator of nicotine reinforcement) and nicotine self-administration. Rats can be trained to self-administer nicotine by pressing a lever to receive a nicotine dose through an indwelling IV cannula. Such a preparation serves as an animal model of smoking. In rats vaccinated over several months and then given access to nicotine, the acquisition of nicotine self-administration was reduced by 40%. In rats already self-administering nicotine, subsequent vaccination substantially reduced self-administration (34) (Fig. 4). Rats did not attempt to overcome the vaccine by self-administering more nicotine to compensate for reduced nicotine delivery to brain. Vaccination also attenuates the reinstatement of nicotine self-administration (the return to self-administration after abstinence triggered by exposure to nicotine), which serves as a model of relapse (36). These data support a potential therapeutic or preventive role for vaccination. Three nicotine vaccines have completed phase I to II clinical trials (16,20). NicVax is nicotine linked to recombinant Pseudomonas aeruginosa exoprotein A with alum adjuvant. In a phase I trial, doses of 50 to 200 mg NicVax administered at 0, 4, 8, and 24 weeks were well tolerated, with transient discomfort

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Figure 4 Immunization reduces nicotine self-administration (NSA) in rats. (A) Acquisition of NSA. Rats were first vaccinated to achieve high antibody titers, then trained to self-administer nicotine over three weeks. For the first week, rats had to press a lever once to receive a dose of nicotine (FR1), the second week twice (FR2), and the third three times (FR3). Vaccinated rats acquired NSA to a lesser extent than controls. (B) Maintenance of NSA. Rats were first trained to self-administer nicotine, and were then immunized with vaccine doses every three weeks. NSA declined after the final vaccine dose. Abbreviations: NSA, nicotine self-administration. Source: From Ref. 35.

Figure 5 Serum antibody concentrations in smokers immunized with a nicotine vaccine (arrows). Antibody concentrations were highest approximately one week after each dose and declined when immunization was stopped, even though most subjects continued to smoke. Source: From Ref. 16.

subjects with the highest third of antibody titers compared to placebo (57% vs. 31%) (M. Bachman, personal communication). While all of these results are preliminary, they appear to be consistent in suggesting some efficacy of vaccination for smoking cessation when high antibody titers are present. All vaccines were well tolerated.

Cocaine at the injection site or minor systemic symptoms such as muscle aches. Serum antibody concentration ranged from 0.002 to 0.13 mg/mL, overlapping the range known to be effective in rats (Fig. 5). Subjects were not asked to quit smoking, since this was not an efficacy trial. However, significantly more subjects in the high NicVax dose group (which also had the highest antibody levels) than in the low dose group spontaneously quit smoking for a period of 30 days at some time during the trial (6/16 vs. 2/23 subjects). There was no evidence of compensation; expired air carbon monoxide concentrations (a measure of smoke intake) did not increase in the vaccinated subjects. TA-NIC vaccine is nicotine conjugated to recombinant cholera toxin subunit B with alum adjuvant. In an unpublished phase I trial, subjects received vaccine at 0, 2, 3, 4, 8, and 12 weeks and a booster dose at 32 weeks (C. Bunce, personal communication). They were invited to quit smoking at 12 weeks. The quit rate at 12 months was higher in the highest dose vaccine group compared to controls (38% vs. 8%, 6/16 vs. 1/12 subjects) but statistical analysis is not available and the number of subjects was small. Nic-Qb vaccine is nicotine linked to a virus-like particle, which was studied in 340 smokers wanting to quit (20). Subjects received five monthly injections of various doses of vaccine. Quit rates at 26 weeks did not differ among the doses tested, but a significantly higher quit rate was found in the

Overview Approximately two million people in the United States abused cocaine in 1998 (37). Behavioral interventions are helpful in treating cocaine addiction, but there are currently no approved medications for this disorder (38). Single and daily cocaine doses are considerably higher than those of nicotine. Even with a single dose, the calculated binding capacity of antibody in vaccinated animals is greatly exceeded (Table 1). Nevertheless, the studies outlined below demonstrate considerable efficacy of vaccination in reducing cocaine effects, illustrating the general principle that drugspecific antibodies can alter drug distribution even when binding capacity is greatly exceeded. Vaccines Linkers for cocaine immunogens have been placed either distant from major sites of metabolism (norcocaine derivatives) (6), or at the methyl ester group to protect it from spontaneous degradation and generation of the major metabolite benzoyl ecgonine (4,39). Both strategies elicit antibodies with high specificity for cocaine compared to its metabolites. A vaccine has also been developed using an anti-idiotype antibody, which mimics the configuration of the cocaine molecule as the immunogen (40). It is not clear whether this approach offers any advantage over use of a conjugate vaccine.

Chapter 89: Immunotherapies To Treat Drug Addiction

Efficacy Passive immunization with cocaine-specific antibodies reduced early cocaine distribution to brain in rats by 25% to 70%, and reduced locomotor activity and stereotypic behavior following a single cocaine dose (4,6). Vaccination also reduced cocaine self-administration in a variety of models (6,23,41,42). As with nicotine vaccines, increasing the cocaine dose did not reliably overcome the suppression of self-administration. Vaccination also reduced the reacquisition (23) and reinstatement (42) of cocaine self-administration in rats, which had been previously taught to self-administer the drug, a model of relapse prevention. In aggregate, these data provide strong evidence that vaccination reduces cocaine self-administration to a quantitatively important extent and in a manner related to serum antibody concentration. In contrast to nicotine, vaccination against cocaine does not appear to alter cocaine’s elimination half-life (6). Accumulation of drug with resulting saturation of antibodies may therefore be less of a problem with cocaine than with nicotine. A conjugate cocaine vaccine consisting of cocaine linked to recombinant cholera toxin subunit B has completed phase I and early phase II clinical trials of immunogenicity, safety, and efficacy. No important adverse effects have been found in these studies and the vaccine has shown a reduction in cocaine effects during human laboratory cocaine administration studies and cocaine use in outpatient studies (43–45). The first outpatient study found that subjects receiving five 400 mg doses of vaccine had a significantly higher mean antibody titer response as compared to four 100 mg doses (2000 vs. 1000 antibody units, p < 0.05). The 400 mg/dose group was also more likely to maintain cocaine-free urines than the 100 mg/dose group (p ¼ 0.002). Most patients reported an attenuation of cocaine’s usual euphoric effects from the vaccination (63% in the 100 mg/dose and 100% in the 400 mg/dose groups). A second human laboratory study found that five subjects who developed higher peak antibody levels after vaccination using a cutoff of 1000 antibody units showed significantly greater reductions in cocaine induced subjective effects than the five ‘‘poor antibody responders’’ who had peak levels below 1000 antibody units. These outpatient high peak antibody responders also reported a 50% reduction in their cocaine use compared to the five poor antibody responders. A larger phase II placebo-controlled and blinded, randomized outpatient clinical trial in 114 methadone maintained cocaine abusers found that a 50% reduction in baseline cocaine use occurred twice as often among those getting active vaccination than among those getting placebo.

Phencyclidine Overview PCP is structurally and pharmacologically related to the dissociative anesthetic ketamine. Abuse is uncommon but important in particular geographical areas. Adverse effects generally consist of acute toxicity from inadvertent overdose and may be severe, but addiction may also occur. Apart from its medical importance, the study of PCP abuse has shed light on the potential role of passive immunization as a treatment strategy. Vaccines A PCP immunogen consisting of PCP conjugated to bovine serum albumin (19) has been used for the production of monoclonal antibodies. In contrast to vaccines for nicotine or

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cocaine, which aim to produce highly specific antibodies, the intent of this PCP immunogen was to elicit poly-specific antibodies that would also bind structurally related drugs that might be abused in place of PCP. This was accomplished by placing the linker distant from the structural features that are conserved among the PCP analogues of interest (46,47) The monoclonal antibody most extensively studied has a Kd of 1.3 nM for PCP. Efficacy Passive infusion of monoclonal PCP antibodies or antibody Fab fragment (the 50 kDa antigen binding fragment of IgG) to rats after acute or chronic PCP doses markedly reduces the PCP concentration in brain (13,48). Protection of the brain is remarkably long-lasting, persisting for a month after a single IgG dose despite continuous infusion of PCP at a daily rate that exceeds the antibody’s binding capacity for drug. Monoclonal PCP antibody also reduces the behavioral toxicity (changes in locomotor activity, posturing) of PCP in rats (46,49). Like the pharmacokinetic effects, behavioral protection is long lasting and is observed up to several weeks after a single passive infusion of antibody (50). Efficacy is apparent even when the antibody dose is equivalent to just 1% of the molar PCP dose (51). PCP antibodies also reverse overt PCP toxicity when they are administered after a toxic PCP dose, and so could be useful in treating overdose (46,49). The considerable monoclonal antibody doses used (up to 0.4 g/kg) have shown no adverse effects.

Methamphetamine Methamphetamine is a stimulant drug with effects similar to those of cocaine but a much longer duration of action. Methamphetamine use in the United States has increased dramatically over the past 10 years, and is associated with considerable medical and social morbidity. Monoclonal antibodies against methamphetamine have been developed to study passive immunization. Passive immunization of rats with a high affinity (Kd ¼ 11 nM) monoclonal methamphetamine antibody reduces methamphetamine self-administration (25). With higher methamphetamine doses (lower antibody:drug ratio), self administration paradoxically increases, indicating compensation and suggesting that providing an adequate antibody dose is critical for optimal efficacy. Methamphetamine antibodies also protect against the increases in blood pressure and heart rate, and locomotor activation (horizontal activity) induced by a high dose of methamphetamine (52,53) (Fig. 6). Passive immunization may therefore be of use for treating methamphetamine toxicity as well as addiction.

Catalytic Antibodies In contrast to drug-binding antibodies, catalytic antibodies are intended to hasten drug metabolism and thereby blunt the drug’s effects. Enhanced metabolism could, if rapid enough, reduce initial drug distribution to brain, and could also prevent the accumulation of drug with repeated dosing. This approach is best suited to drugs that can be inactivated by one-step metabolic processes to an inactive metabolite, such as the hydrolysis of cocaine (54). Cocaine is metabolized in humans primarily to two major metabolites, which are not addictive. One of these, ecgonine methyl ester also appears to be relatively free of toxic effects.

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Onset and Duration of Effect Vaccination will likely require multiple injections of immunogen over several months to achieve maximal antibody titers. Vaccination can be initiated while drug use continues, with cessation of drug use attempted once titers are adequate. Passive immunization may find application when a delay of several months is not clinically acceptable.

Compensation It is likely that addicts will be able to overcome the effects of vaccination, if they are determined to do so, by using higher drug doses. Thus vaccination, like all other medications for drug abuse, will be most successful in highly motivated patients. Fortunately, most cigarette smokers want to quit and are unlikely to purposely sabotage their own efforts. Cocaine or methamphetamine addicts who enter treatment are also motivated to quit, as illustrated by the substantial abstinence rates achieved with behavioral counseling alone. In addition, it does appear that substantial drug doses are needed to overcome the effects of immunotherapy. Figure 6 Passive immunization reduces methamphetamineinduced locomotor activity. In the overdose model, rats were pretreated with methamphetamine (arrow) and then given monoclonal methamphetmaine-specific antibody (arrowhead). In the pretreatment model, rats were first given monoclonal antibody and then methamphetamine. Shading indicates the time period of the duration of drug action above saline-induced (baseline) locomotor activity following buffer or mAb treatments. In both models, antibody reduced total locomotor activity. Source: From Ref. 52.

Vaccination as an Adjunct to Behavioral Treatment As a general rule, drug therapies for addictions are most successful when used in conjunction with behavioral counseling and any necessary psychiatric or social services. It is likely that vaccination, even if effective, will be similar in this regard and should be viewed as an adjunct to counseling.

Combination of Vaccination with Other Therapies

Cocaine hydrolysis to ecgonine methyl ester is catalyzed by the plasma enzyme pseudocholinesterase and proceeds via a shortlived transition state, which is stabilized by the enzyme. An antibody to this transition state could similarly stabilize the transition state and serve as a catalyst (54). Because the transition state is short-lived, it cannot be used as an immunogen, but a stable transition state analog used in this manner can elicit antibodies that markedly speed the degradation of cocaine (55–57). One such monoclonal antibody has been shown to reduce acute cocaine toxicity and cocaine self-administration in rats (41,57). The relative merits of this approach compared to cocaine-binding antibodies are not yet clear.

CLINICAL ISSUES Importance of Antibody Concentration in Serum Vaccination is effective only insofar as sufficient antibody is present to bind drug. For all of the drugs discussed above, antibody dose or concentration is a critical determinant of efficacy. Antibody concentrations elicited in experimental animal studies of drug abuse vaccines (0.05% to 2% of total IgG) have been higher than those typically elicited in humans after vaccines for infectious diseases (1% of total IgG, and often much less) (58,59). In addition, variability in antibody levels achieved after vaccination is substantial. Developing suitable immunogens, adjuvants, and vaccination schedules will therefore be important to the success of this approach. Passive immunization, where antibody dose can be controlled, may prove helpful as an adjunct or alternative to vaccination.

Because its mechanism of action is distinct, it is possible that vaccination will have additive effects with other medications for treating drug abuse. It is also possible that vaccination will target some aspects of addiction better than others and benefit from medications that complement its actions. For example, immunotherapy reduces drug reward and reinforcement that occur when drug is administered, but immunotherapy is not expected to directly affect the craving for drug that occurs when drug is no longer present, and consequently cannot be impacted by antibody. Because craving often leads to relapse, drugs that reduce craving (e.g., bupropion for nicotine addiction) might enhance the efficacy of immunotherapy.

Safety No important adverse effects have been observed to date with either passive or active immunization for drug addiction. However, experience with such vaccines in humans is quite limited. It will be important, in particular, to confirm the specificity of the antibodies elicited to assure that they do not bind endogenous compounds.

ETHICAL ISSUES Since mental illness can interfere with one’s ability to appreciate what vaccination for addiction would mean, and addictions themselves are mental disorders, these vaccines introduce ethical complications. Many ethical complications are shared with long acting depot medications for addictions such as depot naltrexone for alcohol or opiate dependence (60). Like depot medications, vaccine effects persist or remain detectable for a limited but relatively long time period. Active immunizations will attenuate the abused drug for at most a few months,

Chapter 89: Immunotherapies To Treat Drug Addiction

not a lifetime like typical vaccines against viruses or bacteria. The ethical issues are due to particular groups and settings for use. A vaccine might be used in prison or in a residential drug treatment facility or imposed by another party: parents on a child; employer for employment; the government in many guises. Vaccination may be a condition of parole for substance abuse, and it is accepted grudgingly. Does the drug itself being illegal or legal like nicotine influence consent? Suppose its manufacturer is marketing it aggressively, perhaps directly to consumers? A particularly complex ethical situation is parents wanting to vaccinate their children against abused substances (61–63). Physicians need help and manufacturers need control of their promotions for such off-label prevention protocols. Parents seeking to vaccinate their children against substance abuse will likely also spark a debate over the limits of good parenting. A well-considered ethical decision will have to attend to the facts (61). How effective is the vaccine? Is it safe? What is known about other young persons’ reactions to it? Do they substitute other risk behaviors for using the drug, or does the vaccine reduce their overall risk? Some parents live in communities where drug use, violence, and addiction are a realistic danger for adolescents. The possible unintended but anticipatable medical, social, and legal consequences of vaccination can be complicated by the child’s relationship with his/her parents. Has the child been trustworthy and able to resist the enormous peer pressure to do unwise behavior? Instead of a vaccine, might it be preferable to extract a promise not to use the drug to reinforce mutual trust rather than the need for protection against his/her own foolish choices. This decision reflects the family’s core values (62). Large-scale policy options could encourage vaccine use by subsidizing them or requiring insurers to cover their cost. More radically, a city might have a mass vaccination program intended to dry up the market for these drugs. Like herd immunity, if a sufficient percentage of the population is vaccinated, the infection will not spread; vaccinate enough of the community against cocaine, and the dealers will move on to more lucrative markets. In summary, the ethical implications of vaccination against abused substances range from individualized family life to massive public health strategies, and we are just beginning to struggle with these issues.

SUMMARY Vaccines or monoclonal antibodies to treat drug abuse are being studied for several major classes of addictive drugs. Animal data are encouraging, and initial clinical trials appear promising but further clinical trials are needed to confirm efficacy. The main action of vaccines is to elicit antibodies that bind drug and reduce or slow its distribution to the target organ, the brain. Vaccination blocks some of the behavioral effects of nicotine, cocaine, methamphetamine, or PCP even when drug doses substantially exceed the antibody’s drugbinding capacity. Efficacy is closely correlated with antibody titer or concentration in serum, and achieving and maintaining adequate titers will be a major factor in determining whether these vaccines are clinically useful. It is likely that addicts can overcome the effects of vaccination by using larger drug doses, so that vaccination will be most effective for individuals who are highly motivated to quit, and when used in conjunction with counseling. Addiction vaccines have proven safe in

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animals and in limited experience in humans, but a larger clinical experience is needed to establish safety and suitability for clinical use. The use of vaccination in combination with other medications for drug addiction, to enhance efficacy or achieve a greater spectrum of effects, may be possible.

ACKNOWLEDGMENTS Supported in part by NIDA grants DA-10704, DA015668, P50DA13333 (PRP), K05-DA0454 (TRK), and P50-DA18827, and Department of Veterans Affairs, VISN 16, Mental Illness Research, Education and Clinical Center (MIRECC), and VA National Substance Use Disorders Quality Enhancement Research Initiative (QUERI).

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Index A1-ARF interaction, 275 AAV. See Adeno-associated virus (AAV) Ab-producing B cells, 221 ACAM2000, smallpox vaccine, 838 ACIP. See Advisory Committee on Immunization Practices (ACIP) Acquired immunodeficiency syndrome (AIDS), 791, 830–831 ACTG. See AIDS Clinical Trial Group (ACTG) Acute flaccid paralysis (AFP) , 155 Acute lower respiratory tract infection (ALRI), 37 Acylated quinoline-pyrazine, 808 AD. See Alzheimer’s disease (AD) Adaptive immunity, 169–174 control and innate functions of DCs, 327 ADCC. See Antibody-dependent cell-mediated cytotoxicity (ADCC) ADE. See Antibody- dependent enhancement (ADE) Adeno-associated virus (AAV), 589 Adenovirus recombinants vaccine vectors, 352–358 biology, 352–353 replication-competent, 353–355 replication-defective, 354–356 Adenovirus vector serotypes, 356–358 Adjuvanted influenza vaccine, 288 Adjuvanticity, mechanisms, 277–278 Adjuvant MF59 clinical experience with, 288–289 combination with immunopotentiators, 289 composition, 287–288 emulsion, 286–290 manufacturing process, 288 mechanism of action, 287–288 perspectives on use, 290 preclinical experience with, 288 use in prime/boost settings, 289–290 Adjuvants, 250 as ADP-ribosylating enterotoxins, 274 classes of, 251 definition, 250 general principles for actions, 274 in humans. See Experimental adjuvants in humans intranasal adjuvants CTA1-DD, 408 in licensed vaccines for humans, 252–254 mechanisms of action, 250–252 regulatory issues, 252 safety, 252 skin adjuvants, types of bacterial ADP-ribosylating exotoxins (bAREs), 417–418 CpG motifs, 418 imiquimod, 418 vectors, enhancing immunity, 278 Adoptive immunotherapy in EBV-related diseases, 649–650 ADP-ribosylating enterotoxins, 274 ADP-ribosylation mechanism, 274–275 Advanced market commitments (AMC) AMC pilot for pneumococcal conjugate vaccine AMC and post-AMC terms, recommendation of, 76 impact of the AMC, expected, 76 valuation methodology, 75

[Advanced market commitments (AMC)] working of, 74 Advisory Committee on Immunization Practices (ACIP), 430, 574 Aedes aegypti, 559, 566, 593. See also Dengue Aedes albopictus, 559, 566 Aerosols, vaccines delivered as small-particle dry powder aerosol measles vaccine, 409 dry powder aerosol tuberculosis vaccine, 409 liquid aerosol measles virus as vaccine, 408–409 AFP. See Acute flaccid paralysis (AFP) African Region Vaccine Regulatory Forum (AVAREF), 128 African Union (AU), 156 Ag85B-ESAT6 fusion molecule, 525 Ag85B-TB10.4 fusion molecule, 526 Age-dependent limitations of immune responses, 228 Age-related diseases, 239 Aging, 234 AIDS Clinical Trial Group (ACTG) 076 regimen, 60 Airborne particulate classifications, 148 Albendazole, 830 Alfalfa mosaic virus, 309 Allergen-specific immunotherapy, 267 Allergy vaccines, 265–266 Alphaherpesvirus, 235 Alphaviruses, 362–364 ALRI. See Acute lower respiratory tract infection (ALRI) Aluminum hydroxide, 252 Aluminum phosphate, 252 Aluminum salts, 250, 251, 750 Alzheimer’s disease (AD), 420–421, 962 animal models of, 964 clinical and pathological features, 962 genetic factor in development of, 963–964 immunization, treatment by, 964 humans, immunization of, 966–967 studies in animals, 964–966 pathogenesis, role of Ab protein, 962–963 pathological lesions of, 962 VLP-based vaccine against, 302–303 AMC. See Advanced market commitments (AMC) AMC pilot for pneumococcal conjugate vaccine AMC and post-AMC terms, recommendation for, 76 impact of the AMC, expected, 76 valuation methodology, 75 Amebiasis. See also Entamoeba histolytica acquired immunity, 823 Gal/GalNAc vaccination, 826 impact, 820 innate and acquired immune response, 823 intestinal amebiasis, 826 pathogenesis, 820–822 American Academy of Family Practice (AAFP), 430 Amyloid precursor protein (APP), 962–963 AN-1792, AD vaccine, 966 Ancylostoma caninum, 831–833 Ancylostoma ceylanicum, 830–832 Ancylostoma duodenale, 830–831 Animal hookworm infections immunology, 831–832

Anthrax clinical manifestations of, 851 immunity to humoral response, 851 live attenuated spore vaccines, 852–853 PA-based vaccines, 852 pathophysiology of, 851 toxins, 851 virulence determinants of, 851 Anthrax vaccine cell-free, 853 clinical trials, 858 delivery systems for intranasal vaccination, 854–855 transcutaneous vaccination, 855 efficacy studies, 857–859 live. See Live anthrax vaccines plasmid DNA vaccines, 856–857 recombinant viral vectors as, 856 in United States, 853 Anthrax vaccine adsorbed (AVA) adding CpG to, 853 characterization of, 853 efficacy of, 857–858 schedule and route of, 858 vs. recombinant PA–based vaccines, 853 Anthrax vaccine precipitated (AVP), 853 Anthrax virus, 352, 363 Antiamoebic vaccine candidates, 824 Antibody-dependent cell-mediated cytotoxicity (ADCC), 175, 559, 928 Antibody- dependent enhancement (ADE), 595 Anti-DEC-205 with 33D1 antibody targeting, 333–334 hybrid antibodies as vaccines, 330–331 Antigen delivery systems. See also Vaccine adjuvants aluminum salts, 286 emulsion adjuvant MF59, 286–290 recent developments in, 284–294 Antigen expression chromosomal strategies, 379 engineering immune responses, 378 in transgenic plants, 309 Antigenic protein expression in plants, 306–307 Antigen-presenting cells (APCs), 168, 238, 263, 284–285, 315, 415 Antigen processing by MHC molecules, 172–173 nonclassical MHC Class I and other molecules in, 173–174 by T cells, 172 use of epitopes in, 174 Antigen-specific B-cell ELISPOT, 223 Anti-lipopolysacharide (LPS) antibodies, 825 APC. See Antigen presenting cells Apo-lipoprotein E (ApoE), 964 APP. See Amyloid precursor protein (APP) Ascariasis, 830 AS04 vaccine, 253–254 Attenuated dengue viruses, 13 AU. See African Union (AU) Autoantigen-specific vaccination, 969–970, 976 Autoclave-killed vaccine, 795 Autoimmune adverse effects vaccine administration and, 244 vaccine-related, 245–246 MS development, 245

994

Index

Autoimmune disease, and vaccination, 969 atherosclerosis, 977 chronic inflammatory disorders, 976 insulin resistance and type 2 diabetes, 977 multiple sclerosis (MS), 975–976 nonobese diabetic (NOD) mouse, studies on, 971 DNA vaccination, 972 mucosal vaccination, 971–972 systemic vaccination, 972, 973 obesity, 976–977 rheumatoid arthritis (RA), 975 type 1 diabetes (T1D), 970–971 vaccination strategies, 969 autoantigen vaccination, 969–970, 976 vaccination trials in humans, 972–975 Diabetes Prediction and Prevention Project (DIPP), 974–975 Diabetes Prevention Trial (DPT)-1, 972–974 intranasal insulin trial I (INIT I), 974 NIH TrialNet, 974 Autoimmune diseases, 242 autoimmune encephalomyelitis, 175 infectious agents as trigger of, 234 vaccine-attributable, 245 Autoimmune responses, 242 Autoimmunity induced by infectious agents, mechanisms, 243 bystander activation, 244 molecular mimicry hypothesis, 243 regulatory T cells, 243 self-antigens, enhanced presentation of, 243 vaccine-associated risk of, 244 Autologous CTL-based therapy, 649–650 Autolysin, 484 Avian influenza, 344 Bacille Calmette-Gue´rin (BCG), 3, 13, 83, 123, 265, 353, 397, 399 auxotrophs and rBCG auxotrophs, 521–522 efficacy of, 516 Bacillus anthracis, 3, 17, 18, 418, 851 Bacillus anthracis vaccine, 267 Bacterial ADP-ribosylating exotoxins (bAREs), 253, 417–418 Bacterial DNA (CpG sequences), 286 Bacterial surface polysaccharides, 489 Bacterial vaccines, early inactivated whole-cell cholera vaccines, 4 typhoid vaccine, 4 Bacteroides thetaiotaomicron, 199 BALT. See Bronchus-associated lymphoid tissue(BALT) BAREs. See Bacterial ADP-ribosylating exotoxins B-cell epitopes, 171 memory, 221 BCG. See Bacille Calmette-Gue´rin (BCG) Bean yellow dwarf virus, 309 Bell’s palsy, 544 b-secretase, 967 BILHVAX, clinical efficacy of, 811 Biologic Licensing Application (BLA), 26, 116, 118 Biologics Control Act, 122 Biomarkers for vaccines, 118 Bio-Plex Luminex1 platform assay, 891 BLA. See Biologic Licensing Application (BLA) Blocking antibody, 201

Blood-stage malaria vaccines, 753, 757 in clinical development apical membrane antigen 1 (AMA1), 754–755 combination blood-stage antigen vaccines, 756–757 erythrocyte-binding antigen 175 (EBA175), 756 GMZ2, 757 merozoite surface protein 1 (MSP1), 755–756 merozoite surface protein 2 (MSP2), 756 merozoite surface protein 3 (MSP3), 756 PfCP-2.9, 757 serine repeat antigen 5 (SERA), 756 development of, 753–754 B Lymphocytes, 170, 239 memory, 171 responses, induction, 170 Booster vaccinations in adults, 240 Booster vaccine, 520 Ag85B-ESAT6 fusion molecule, 525 Ag85B-TB10.4 fusion molecule, 526 85B-ESAT-6 hybrid vaccine, 522 MTB72f vaccine, 526 MTB 30 kDa major secretory protein, 522 MVA85A, 526–527 Bordetella pertussis, 175, 437 Borrelia burgdorferi, 133, 134 Botulinum toxin, 363 Bovine leukemia virus, 342 Bovine parainfluenza virus, 366 Bovine viral diarrhea virus, 365 Braun’s lipoprotein, 320 Bromoviruses, 309 Bronchopneumonia, 872 Bronchus-associated lymphoid tissue (BALT), 405 Brucella, 364 Brucella melitensis, 17 Bubonic plague, 872–873 Bystander activation, 244 Bystander suppression, 969 CAIV. See Cold-adapted influenza vaccine (CAIV) Calcium phosphate, 252 Campylobacter jejuni, 242, 243, 534 Canarypox, 344, 355 HIV vaccine, 344 virus, 637 Cancer. See also Cancer-specific vaccines ex vivo-derived antigen-loaded in therapy, 329 heterologous primeboost technology, 402 plant-derived vaccines, 310–311 Cancer antigens cancertestis antigens, 929 self-antigens, 929 tumor-selective antigen, 929 tumor-specific antigens, 929 Cancer-specific vaccines antigen-specific vaccines peptide vaccines, 940 cancer antigens cancertestis antigens, 929 self-antigens, 929 tumor-selective antigen, 929 tumor-specific antigens, 929 cancer vaccine efficacy, enhancement of, 945–946 ex vivo antigen-loaded dendritic cell vaccines, 941–942 heat shock protein-based vaccines clinical trials, 942

[Cancer-specific vaccines] immune surveillance of cancer epidemiologic studies of Immunodeficiencies patients, 930 tumorigenesis studies in genetically manipulated mice, 931 immune tolerance and immune evasion dendritic cell (DC) , activation, 932 induction of T-cell tolerance, 931 immune tolerance and immune evasion, 932 innate immunity, epithelial immunity, and tumor immune surveillance, 931 oncogenic pathways, 934–935, NF–k B pathway (IKK) Braf pathway, 935 STAT3 pathway, 934 oncogenic pathways, 934 prostate cancer vaccines, comparison of two, 943–945 regulatory T cells and cancer, 933–934 tumor microenvironment, immunologic characteristics of, 937–938 tumor vaccines, genetically modified GM-CSF Gene-transduced tumor vaccines, 939 vaccine vectors engineered bacteria, 943 engineered viruses, 942–943 whole-cell tumor vaccines, 938–939 Cancer vaccines, 265 Cancer vaccine vectors engineered bacteria, 943 engineered viruses, 942–943 Canine distemper virus, 344 irradiated larval vaccine paradigm, 832 parvovirus, 309 Capillary sequencing, 15 Capsular polysaccharides (CPS), 691 ‘‘Catch-up’’ dengue vaccine campaigns, 598 CBER. See Center for Biologics Evaluation and Research; Center for Biologics Evaluation and Research (CBER) CBER-generated confirmatory lot, 118 CCR7. See Chemokine receptor 7 CD27þ B cells, 239 CDC. See Centers for Disease Control and Prevention CD8þ cytotoxic T lymphocytes role in measles infection prevention, 580 CD8þ cytoxic T lymphocytes, 534, 635, 969 CD4þ helper T cells, 534 CD1-restricted antigens, 17 CD8 T cells biological functions, 517, 518 stimulation of, 518 CD4þ T-helper cells, 238, 239 Cell-cycle inhibitor p16INK4A, 237 Cell-free anthrax vaccine, 853 Cell-mediated immunity in measles infection prevention, 580 Center for Biologics Evaluation and Research (CBER), 116 guidance to facilitate vaccine clinical development, 119 international collaborations, 119 regulatory authority communication for product development, 118 licensure, 117–118 managing review process, 118 post-market, 118 pre-market, 117

Index [Center for Biologics Evaluation and Research (CBER)] regulatory authority, implementation of communication with CBER, 118 licensure, 117–118 managed review process (MRP), 118 post-market, 118 pre-market, 117 role in product development, 119 Center for Disease Control and Prevention (CDC) , 222, 597, 838, 885 CervarixTM, 254 CFR. See Code of Federal Regulations (CFR) CFs. See Colonization factors CFTR. See Cystic fibrosis transmembrane conductance regulator CGMP. See Current Good Manufacturing Practice Chemistry, manufacturing, and controls (CMC) testing, 116 Chemokine receptor 7, 238 Chemokines, 176, 177 Childhood immunization programs. See Immunization schedules Chimeric flavivirus vaccines. See Flavivirus Chimeric rBRSV/HRSV, 627 Chimerivax vaccines ChimeriVax-DEN clinical trial of, phase I, 566 ChimeriVax-JE safety and efficacy profile, 563–564 ChimeriVax-WN clinical trial of, phase I, 566 Chimpanzee model, HCV infection, 605 natural immunity against HCV, 607 vaccination with adenoviral vectors, 614 Chiron vaccine, 660–661 Chitosan, 408 Chlamydia, 175 Chlamydia pneumoniae, 17, 81 Chlamydia psittaci, 737 Chlamydia trachomatis, 319, 364, 737. See also Chlamydia trachomatis infection Chlamydia trachomatis, 418 Chlamydia trachomatis infection future perspectives, 742–743 immune responses to CD4þ and CD8þ T cells role in, 739 immune responses to, vaccines for MOMP vaccines, 740 multisubunit vaccines, 742 non-MOMP vaccines, 742 Chlamydia trachomatis vaccines MOMP vaccines multisubunit vaccines, 742 non-MOMP vaccines, 742 MOMP vaccines, 740 Chloroplast transformation, 310 Cholera disease, 308, 793 immune mechanisms adaptive immune mechanisms, 508 antitoxic cholera immunity, 508 colonization factors and antibacterial immunity, 508–509 innate immunity, 507–508 killed O139 cholera vaccine candidates, 510 killed whole-cell cholera vaccines herd protection killed WC-Only Vaccines, 509–510 killed WC-rCTB Vaccine, 509 reduction of diarrhea, morbidity and mortality live attenuated cholera vaccines recombinant vaccine candidates, early generations of, 511 V. cholerae 638, 512

[Cholera disease live attenuated cholera vaccines] V. cholerae CVD 103-HgR, 511–512 V. cholerae O1 and O139 vaccine candidate V. cholerae PERU-15, 512 vibrio cholerae, causative agent Epidemiology of, 506 virulence factors and pathogenesis, 507 Cholera toxin (CT), 273, 308, 407, 544, 714 Cholera vaccines, 4 Cholera vaccines, oral immune mechanisms in cholera adaptive immune mechanisms, 508 antitoxic cholera immunity, 508 colonization factors and antibacterial immunity, 508–509 innate immunity, 507–508 killed O139 cholera vaccine candidates, 510 killed whole-cell cholera vaccines herd protection, 510 killed WC-Only Vaccines, 509–510 killed WC-rCTB Vaccine, 509 reduction of diarrhea, morbidity and mortality, 510 live attenuated cholera vaccines recombinant vaccine candidates, early generations of, 511 V. cholerae 638, 512 V. cholerae CVD 103-HgR, 511–512 V. cholerae O1 and O139 vaccine candidate, 512 V. cholerae PERU-15, 512 vibrio cholerae, causative agent cholera disease, 506 epidemiology of, 506 virulence factors and pathogenesis, 507 Chromosomal antigen expression, 379 Chronic granulomatous disease (CGD) , 729 CIOMS. See Council for International Organizations of Medical Sciences (CIOMS) Circumsporozoite protein (CSP), 399, 747, 752, 774, 775, 777, 778 Classical Mexican device, (CMD), 408 Class II MHC expression, 176 Class II pathogens, 191–193 characteristics of, 193 course of infection of host with, 192 examples of, 192–193 Class I pathogens, 191, 192 characteristics of, 192 course of infection of host with, 192 Clathrin-dependent endocytosis, 607 Claudin-1 expression, 606 Clinical studies of malaria vaccines adenovirus vectors Ad5 AMA1, 778 AdCh63 ME-TRAP, 778 DNA vectors circumsporozoite protein (CSP) , 776 multiple epitope-thrombospondinrelated adhesionprotein (ME-TRAP), 776 SSP2/TRAP, 776 DNA vectors in prime-boost regimens DNA-MVA regimens, 777 poxvirus vectors MVA ME-TRAP, 777 NVYAC Pf7, 777 prospects of, 778–710 Clinical trials of vaccines, randomized overview of allocation and administration, 43 eligibility, 42 recruitment, 42 surveillance, 43 target population, 42

995

Clostridium difficile, 418 Clostridium tetani, 4 CMD. See Classical Mexican device (CMD) CMI. See Cell-mediated immunity CMV. See Cytomegalovirus (CMV) Code of Federal Regulations (CFR), 27, 28, 29, 116, 119 Cold-adapted influenza vaccine (CAIV), 537–538 adults and, 540–541 attenuation of master donor viruses, genetic basis, 538 children, studies on, 539–540 cold adaptation, process of, 537–538 development of, 537 and elderly population, 541 immune responses, 539 live attenuated viruses, use of, 537 protective efficacy of, 539–541 safety of, 538–539 Colonization factors composition of, 715 ETEC vaccine based on, 716 on human ETEC, 714 immunologically related, 715 CombiHIVvac vaccine, 14 Combination vaccines acellular pertussis vaccine,based, 437–438 regulatory aspects of, 436–437 safety evaluation of, 437 Combined pneumococcal protein vaccine protection against Streptococcus pneumoniae strains, 485 Community-acquired pneumonia infections, 234–236 Community-associated methicillin-resistant S. aureus (CA-MRSA), 728 Comoviruses, 309 Comparative genome hybridization (CGH), 18 Compared agents administration, issues in, 48 allocation, issues in blocked allocation, 47 stratified allocation, 47 Complete Freund’s adjuvant (CFA), 317, 330 Conjugate vaccines, 454 Coronaviruses, 361, 363–364 Corrective action preventive action (CAPA) system, 149 Corynebacterium diphtheriae, 4, 5 Cost-benefit analyses, 108 Cost-effectiveness analysis (CEA), 108–109 Cost-utility analysis (CUA), 109 Council for International Organizations of Medical Sciences (CIOMS), 60, 61, 62, 63, 64 Cowpea mosaic virus, 309 Cowpox virus, 340 Coxsackievirus and adenovirus receptor (CAR)-binding receptor, 357 CpG adjuvants general utility, 263–264 safety of, 267–268 synergy with, 264–265 testing infectious disease vaccines with, 266 as vaccine adjuvants, 267 and vaccine hyporesponsiveness, 264 veterinary applications, 266–267 CpG-allergen conjugates, 266 Critical path opportunities list, 118 CRM. See Cross-reactive material Cross-protective immunity, 607 Cross-reactive material, 219 Croup, 620 CSP. See Circumsporozoite protein (CSP) CT. See Cholera toxin (CT) CTA1-DD adjuvant, for enzymatic activity, 276–278

996

Index

CTA1-R7K-DD molecule, 278 CTB-adjuvanted immunizations, 274 CT holotoxin, 276 CTL. See Cytotoxic T lymphocytes CTL response induction with EBV vaccine formulations, 648 C-type lectin receptor (CIRE), 334 Culex mosquitoes, 559, 566 Current Good Manufacturing Practice, 116 Cutaneous leishmaniasis, 792–793, 795–797, 799 Cutter incident, 122 CVD 1902 and CVD 1903 (live oral typhoid vaccine), 502–503 CVD 908-htrA (live oral typhoid vaccine), 30, 500 CVD 908 (live oral typhoid vaccine) licensure, 500 phase I clinical trials, 499–500 CVD 909 (live oral typhoid vaccine), 500 Cyanoacrylate skin surface stripping, 542 29-kDa cysteine–rich E. histolytica antigen, 823–824 Cystic fibrosis transmembrane conductance regulator, 275 Cytokines, 175, 256 Cytomegalic inclusion disease (CID), 633 Cytomegalovirus (CMV), 239, 289–290, 363, 589, 633 Cytotoxic T lymphocytes (CTLs), 231, 251, 264, 534, 544 DALYs. See Disability-adjusted life years (DALYs) Data and safety monitoring boards (DSMBs), 39, 50, 135 Data monitoring committees (DMCs), 135 DCs. See Dendritic cells DC-SIGN (CD209), 334 DCVMN. See Developing countries vaccines manufacturers network (DCVMN) DCVRN. See Developing Countries Vaccine Regulators Network Deceptive imprinting, 193, 200–201 characteristics of, 193 Declaration of Helsinki, 60–64 Declaration of Talloires, 155 Dectin-1, 327, 334 Dectin-2, 327, 334 Delayed-type hypersensitivity, 175 Dendritic cells (DCs), 167–169, 239, 273, 285, 315 adjuvants for immune responses, 329 antibodies targeting antigens to, 332–334 anti-DEC-OVA targeting to, 332 exposed to antigens ex vivo, 329 ex vivo-derived antigen-loaded, 329 innate functions, 327 mediated tolerance and maturation, 328–329 positioning and homing, 327 receptors and expression, 327–328 targeting DNA vaccines, 335 for targeting protein vaccines, 334–335 targeted vaccines, 335 targeting of tumor cell vaccines to, 329–330 tolerance and immunity outcomes after antigen targeting to DEC-205, 330–331 vaccines based on, 327–335 composed of, 329 proteins targeting to maturing, 330–333

Dengue, 593 fever, 363 immune response to, 594–595 and pathogenesis of DHF/DSS, 594–595 protective immunity, issues of, 594 and role for ADE, 595 vaccine for. See Dengue vaccine, development of world distribution of, 593, 594 Dengue (DEN) virus, 284, 593–594 chimerivax vaccines ChimeriVax-DEN, 565–566 dengue intertypic chimeras, 560–561 serotypes of, 593, 594 structure and replication of, flavivirus, 557–559 Dengue hemorrhagic fever (DHF), 559. See also Dengue Dengue shock syndrome (DSS), 559. See also Dengue Dengue vaccine, development of, 594 history of, 595 inactivated vaccines, 597 LAV vaccines. See Live attenuated virus (LAV) vaccines, for dengue need of, 593 purified inactivated vaccines, 597 recombinant vectored vaccines, 598 subunit vaccines, 597 tetravalent dengue vaccines, 595 unique and complex issues in, 598–599 vaccines, potential uses of, 598 DEN virus. See Dengue (DEN) virus Detoxification strategies of CT and LT, 275–276 Developing countries costs and benefits of Hib vaccines, 469–470 efficacy of protein-conjugated Hib vaccine in, 464–465 Hib disease burden measurment Hib rapid assessment tool (Hib RAT), 467–468 hospital and community-based surveillance, 467 population-based surveillance, 467 Hib vaccine introduction, impediment to, 470 programmatic infant Hib immunization, impact of The Gambia, 468 herd protection effect, 469 Kenya, 468–469 serotype replacement disease, 469 vaccine economics Advanced Market Commitments (AMC), 74–77 development costs in a perfect world, 68–69 global vaccine market, 66 International Finance Facility for Immunization (IFFIm), 73 pricing, 67–68 pull and push mechanism, 73 suppliers of vaccine, 66–67 vaccine production, 69–72 vaccine trials in controversies, current, 61–64 ethical principles and rules, 60 international ethical guidelines for biomedical research with human subjects, 60–61 international regulation for, 60 trial design and implementation, issues related to, 36–38 trial oversight, issues related to, 38–39 trial site preparation, issues related to, 34–36

Developing countries vaccine regulators network, 125, 128 Developing countries vaccines manufacturers network (DCVMN), 83 Developing country manufacturers partnerships among MNCs, 152 vaccine production enhancement, 151 vaccine technological capacity of, 151–152 DHF. See Dengue hemorrhagic fever (DHF) Diabetes Prediction and Prevention Project (DIPP), 974–975 Diabetes Prevention Trial (DPT)-1, 972–974 Diarrheal diseases, 570, 831 Dihydroepiandosterone (DHEA), 543 Dihydrofolate reductase-thymidylate synthase (DHFR-TS) L. major knockout, 794 4-(4,6-Dimethoxy[1,3,5]triazin-2-yl)-4methyl-morpholinium, 222 Diphtheria-tetanus-pertussis (DPT), 465 vaccine, 815 Diphtheria toxoid-conjugated meningococcal ACYW CV vaccine, 444 DIPP. See Diabetes Prediction and Prevention Project (DIPP) Disability-adjusted life years (DALYs), 105 burden of disease in, 790–791 hookworm infection impact, 830–831 Disposables, in vaccine manufacturing, 150–151 DLN. See Draining lymph nodes (DLN) DMAIC (define, measure, analyze, improve, and control) cycle, 150 DMCs. See Data monitoring committees (DMCs) DMTMM. See 4-(4,6-Dimethoxy[1,3,5]triazin2-yl)-4-methyl-morpholinium DNA vaccines, 231, 265, 811–814 adjuvants and cytokines, 388 cellular immune responses, 386–387 clinical studies, 390 encoding MTB Antigen 85A, 523 formulation and delivery, 388 human trials, 389–391 humoral responses, 387 innate immunity, 388–389 live vector delivery, 380–382 mixed-modality vaccination, 388 mucosal and transcutaneous administration, 388 novel DNA constructs, 389 potency, 387–389 prime-boost vaccination, 388 targeting DC receptors, 335 veterinary use, 389 DNA vectors immunogenicity of, 582 Double-stranded RNA (dsRNA), 286 DPT. See Diabetes Prevention Trial (DPT)-1 Draining lymph nodes (DLN), 415 Drug addiction, and immunotherapy, 982 catalytic antibodies, 987–988 clinical issues antibody concentrations, importance of, 988 combination with other therapies, 988 compensation, 988 onset and duration of effect, 988 safety issues, 988 vaccination as adjunct to counseling, 988 cocaine, 986 efficacy of vaccine, 987 and vaccines, 986 ethical issues, 988–989 mechanism of action, 982–984

Index [Drug addiction, and immunotherapy] methamphetamine, 987, 988 nicotine, addiction to, 985 active vs. passive immunization, 985 efficacy of vaccination, 985–986 vaccines, 985 passive immunization, 985 phencyclidine abuse, 987 efficacy of PCP antibodies, 987 PCP immunogen, 987 vaccine formulation, 984 carrier proteins, 984 linkers, 984 vaccine schedules, 984 antibody concentration in serum, 984–985 Dry Patch Iomai dry patch vaccines animal models, testing in, 422–424 clinical studies in humans, 424–426 manufacturing, 421 patch thermostability LT and flu dry patches, 421 Dryvax, 838 DSMBs. See Data and safety monitoring boards (DSMBs) DSS. See Dengue shock syndrome (DSS) DTH. See Delayed-type hypersensitivity DT104 infection, 492 DTP. See Diphtheria Tetanus Pertussis (DTP) DTPa-based vaccines, 229 Early immunization neonatal tolerance by, 229 priming of memory cells, 229 Early-life antibody responses influence of maternal antibodies on, 230 limitations characteristics of age-dependent limitations, 228 duration of, 228 of marginal zone B-cell responses, 228 to polysaccharide antigens, 227 to protein antigens, 227 and quality of antibody responses, 228–229 perspectives for enhancing, 230 of CD4þ and CD8þ vaccine responses, 231 Ebola and Marburg viruses animal models for protection, studies on, 880 guinea pig model, 880–881 murine model, 880–881 disease symptoms, 879 features of, 879, 880 history of infection, 879 human vaccines, development of, 882–883 mortality rates for, 879 natural immunity in humans, 881 nonhuman primates (NHPs), vaccines in, 881 outbreaks of, 879 steps in testing and potential licensure of vaccine, 882 structure and genome, 879–880 zoonotic transmission of, 879 Ebola virus, 284, 352, 356, 358, 363, 366 EBV. See Epstein–Barr virus EBV infection, primary cytotoxic T-cell response, 646–647 LMP protein levels, 646 model of, 647–648 vaccines associated with, 646, 648–649 EBV vaccine formulations and immunotherapy, 648 incorporating EBV latent proteins, 648–649 incorporating lytic protein, 648 opportunities and challenges in, 647

[EBV vaccine formulations] testing in HLA transgenic mice and rhesus monkeys, 648 ECBS. See Expert Committee on Biological Standardization Economic analysis components of, 109–112 sensitivity analyses, 113 used to evaluate vaccines and vaccination programs, 109 Economic equation, innovative mechanisms to change pull and push mechanism, 73 ECTV. See Orthopoxvirus ectromelia virus (ECTV) Edema factor, 851 Edmonston-Zagreb (EZ) measles vaccines, 407 EF. See Edema factor EHEC. See Enterohemorrahagic Escherichia coli Eimeria coccidiosis, 266 EIR. See Entomological Inoculation Rate (EIR) ELI. See Expression library immunization (ELI) ELISA assay, 220, 221 ELISPOT. See Enzyme-linked immunosorbent spot EMEA. See European Medicines Agency (EMEA) Entamoeba dispar, 820–821 Entamoeba histolytica acquired immunity in human, 823 Gal/GalNAc vaccination, 826 pathogenesis, 820–822 adherence, 820–821 cytolytic activity, 821–822 invasion, 821 serum resistance, 822 vaccines, 820–826 candidates, 823–824 oral, 825–826 parenteral, 824–825 Enteric-coated capsule formulation of Ty21a, 498 Enterohemorrahagic Escherichia coli infections with, 493 virulence factor of, 493 Enterotoxigenic Escherichia coli (ETEC), 415, 425 antigenic heterogeneity of, 723–724 diarrhea due to, 714 E. coli E1392-75-2A, and anticolonization immunity, 544, 724–725 incidence of, 714 infection-derived immunity to, 724 multivalent Shigella/ETEC vaccine, 725 pathogenic mechanisms colonization factors (CFs), 714–715 enterotoxins production, 714 LT and ST, 714 target antigens, 723 Entomological inoculation rate (EIR), 758 Env and Gag proteins, 14 Enzyme-linked immunosorbent spot, 221 Eosinophils, 167 Eph receptors, 886 EPI. See Expanded Programme on Immunization Epitope-based vaccines, 582 Epstein-Barr virus, 239, 317, 344, 352 DNA levels and disease stage in NPC, 650 gene expression in EBV-associated diseases, 646–647 immune response to, 651 in nasopharyngeal carcinom, 81 primary infection. See EBV infection, primary Equine arteritis virus, 363

997

Escherichia coli, 16, 18, 25, 26, 51, 273, 407, 447 enterohemorrahagic. See Enterohemorrahagic Escherichia coli enterotoxigenic. See Enterotoxigenic Escherichia coli recombinant Lyme disease vaccine, 306 Escherichia coli O157:H7 outbreak of, 493 risk in children, 493 vaccine development activities against conjugate vaccine for children, 494 veterinary vaccine in cattle, 494–495 ETEC. See Enterotoxigenic Escherichia coli (ETEC) ETEC candidate vaccines CF antigens, 716 evaluation, 720 inactivated. See Inactivated ETEC vaccines live attenuated. See Live attenuated ETEC candidate vaccines rCTB-CF ETEC, 718 ETEC disease epidemiology of, 714 immune responses against CF antibodies, 715 IgA antibody responses, 716 mucosal, 716 systemic and mucosal antibody levels, 715 Ethical Considerations in Biomedical HIV Prevention Trials, 61 Ethical principles of research in developing countries beneficence, 60 justice, 60 respect for communities, 60 respect for persons, 60 European Medicines Agency (EMEA), 430 European Medicines Authority, 119, 128 Exotoxins, 253, 255 Expanded program on immunization (EPI), 122, 579 antigens, interventions and inclusion of new, 96 disease control goals of, 94–96 equipment auto-disable syringe, 93 expansion, of UNICEF role in, 93 ice lined refrigerators (ILR), 92 immunization, improving access to, 97 implementation, evaluation, and oversight, 93 origins of, 91–92 vaccines, introducing new life-saving, 98 Experimental adjuvants in humans cytokines, 256 emulsion-based formulations, 254–255 exotoxins, 255 immunostimulatory oligonucleotides, 256 monophosphoryl lipid A, 255 nonionic block copolymers, 255–256 saponins, 255 Expert Committee on Biological Standardization, 121 Expression library immunization (ELI), 448 EZ. See Edmonston-Zagreb (EZ) measles vaccines FDA. See Food and Drug Administration (FDA), U.S. FD&C Act. See Food, Drug and Cosmetic Act Federalwide Assurance (FWA), 38 Feline infectious peritonitis virus (FIPV), 917 Feline leukemia virus, 344 Fendrix hepatitis B vaccine, 798 Filamentous phage, 815 Filoviruses, 365 FIPV. See Feline infectious peritonitis virus (FIPV)

998

Index

FI-RSV. See Formalin-inactivated RSV Flaviviruses, 361, 363–364 chimerivax vaccines ChimeriVax-DEN, 565–566 ChimeriVax-JE, 562–564 ChimeriVax-WN, 566 diseases and available vaccines dengue virus, 559–560 Japanese Encephalitis virus, 559 West Nile virus, 560 yellow fever virus, 560 flavivirus chimeras chimeras between unrelated flaviviruses, 561–562 dengue intertypic chimeras, 560–561 structure and replication, 557–559 F4/80-like-receptor (FIRE), 334 Fluad1, 288 FLUADTM vaccine, 253 Follicular lymphomas, 310 Food, Drug and Cosmetic Act (FD&C Act), 27, 116 Food and Drug Administration (FDA), U.S., 116 critical path initiatives for vaccine development biomarkers for vaccines, 118 disease models, improved, 119 manufacturing capabilities, 118 licensure of vaccines, 116 regulatory authority, overview of Center for Biologics Evaluation and Research (CBER), 116 Code of Federal Regulations (CFR), 116 Office of Vaccines Research and Review (OVRR), 116 Food and Drug Administration Modernization Act of 1997, 116 Food-delivered vaccines, 307, 311 Foot and mouth disease virus (FMDV), 308–309, 367 Foreign antigen expression, 375–377 toxicity, 377–378 Formalin-inactivated RSV, 622–623 Fowlpox virus vaccines, 344 F protein (PFP) vaccines, 623 Francisella tularensis, 17, 20 Freund’s adjuvant, 824–825 Freund’s incomplete adjuvant (FIA), 287 FWA. See Federalwide Assurance (FWA) GABHS (group A b-hemolytic streptococci) challenge, 679 GACVS. See Global advisory committee on vaccine safety (GACVS) GALT. See Gut-associated lymphoidtissue (GALT) gDPGA capsule. See Poly-g-D-glutamic acid capsule GAP. See GTPase-activating protein GAS pharyngitis incidence of, 675 Gastric inhibitory peptide (GIP), 977 GAS vaccine based on SCPA protein, 674–675 desirable characteristics of, 673 GRA CHO antibodies, 675–676 safety issues, 675 GAVI. See Global Alliance for Vaccines and Immunizations (GAVI) GAVI alliance (GAVI ), 151 GAVI’s independent review committee (IRC), model. See also Global Alliance for Vaccines and Immunization (GAVI) recommendations GAVI’s independent review committee (IRC), model deciding factors for, 103 review process, 103

GBS. See Group B Streptococcus (GBS) disease; Guillan-Barre´ syndrome (GBS) GCP. See Good Clinical Practice (GCP) Geminiviruses, 309 Gene gun device, 542 General Practice Research Database (GPRD), 136 Genes delivery adenovirus biology and vector development for, 352–353 Genome-derived vaccines limitation of, 17 Genomics comparative genomics GBS pan-genome, exploiting, 19 pan-genome, 18–19 functional genomics global genomic profiling using microarrays, 19–20 protein array technology, 20 proteomics, 20 revolution epitope predictions and immunomics, 17–18 reverse vaccinology, 15–17 structural genomics, 20 GFIMS. See Global Framework for Immunization Monitoring and Surveillance (GFIMS) Ghost tangles, 962 Ghrelin, 976–977 GIP. See Gastric inhibitory peptide (GIP) GlaxoSmithKline (GSK) biologicals, 83 GlaxoSmithKline vaccine, 660–661 Global advisory committee on vaccine safety (GACVS), 98 Global Alliance for Vaccines and Immunization (GAVI), 576 country proposal review process GAVI Alliance and Fund Boards, 103 GAVI’s independent review committee (IRC), model, 102 International Finance Facility for Immunization Entities, 104 National Interagency Coordinating Committees, 104 proposals review process, 103 Regional Working Groups (RWGs), 104 secretariat, 104 task teams, 104 working group, 104 GAVI support Civil Society Organization (CSO) support, 102 Health Systems Strengthening (HSS), 102 Immunization Services Support (ISS), 102 new and underused vaccines support (NVS), 102 issues and challenges funds, 106 safety and waste management, 105–106 sustainability, 105 vaccine supply, 105 research and development, 105 strategic goals, 101 Global Framework for Immunization Monitoring and Surveillance (GFIMS), 97 Global Immunization Vision and Strategy (GIVS) strategic area of, 96–97 Global Polio Eradication Initiative (GPEI). See Polio Eradication GM-CSF. See Granulocyte-macrophage colony-stimulating factor GM1-ganglioside, 275 GMP. See Good manufacturing practices

Gonadotrophin releasing hormone (GnRH), 317 Gonococcus virus, 363 Good clinical practice (GCP), 27, 28, 117, 125 Good manufacturing practices (GMP), 147 applications, 148–149 documentation, 149 environmental conditions, 148 equipment used in, 149 facilities, 148 pharmaceutical water, 149 basic components, 147–148 initiatives, 118 GpE2 DNA vaccine, 613 GPEI. See Global Polio Eradication Initiative (GPEI) GPRD. See General Practice Research Database (GPRD) GRA CHO antibodies, 675–676 Granulocyte-macrophage colonystimulating factor, 256, 265 Group A streptococcal (GAS) infections antibiotic prevention of, 673 clinical features of, 672 heteropolymer-induced protection, 678–679 prevalence of, 672–673 serotype-independent protection against, 673 intranasal immunization, 674–675 SC injections of SCPA, 675 Group A Streptococcus (GAS), 316–317, 319 Group B Neisseria meningitidis, 284, 291 Group B Streptococcus (GBS) disease in children, 690 GBS proteins, for use in vaccines, 696 a C protein, 696 b C protein, 696 C5a peptidase, 696 leucine-rich repeat surface protein, 696 pilus-like structures, 696–697 Rib protein, 696 Sip protein, 696 in neonates and young infants, 690 in nonpregnant adults, 691 in pregnant women, 690–691 vaccines for alum as adjuvant, 695 and future directions and issues, 695–696 GBS CPS vaccines, 691 GBS type III CPS-protein conjugate vaccine, 691–692 GBS type V conjugate vaccine, 694 maternal immunization, with III-TT, 695 type II CPS conjugate vaccines, 693 type III-TT vaccine, 694–695 type II-TT/III-TT bivalent vaccine, 694 types Ia and Ib CPS conjugate vaccines, 692–693 GTPase-activating protein, 275 Guillan-Barre´ syndrome (GBS), 52, 242, 534–535 Gulf war syndrome, 134 Gut-associated lymphoidtissue (GALT), 405 HA. See Hemagglutinin (HA) HAART. See Highly active antiretroviral therapy (HAART) Haemophilus influenzae, 13, 26, 36, 41, 67, 81, 92, 98, 101, 219, 234, 418, 474 Haemophilus influenzae type b epidemiology of, 452–453 polysaccharide capsules of, 452 transmission of, 452

Index Haemophilus influenzae type b (Hib) disease burden measurment Hib rapid assessment tool (Hib RAT), 467–468 hospital and community-based surveillance, 467 population-based surveillance, 467 conjugate vaccines, 489 efficacy of conjugate vaccines in, 454 efficacy of unconjugated PRP vaccine in, 454 in England and Wales, 454, 455 herd protection effect, 469 Hib disease burden measurement Hib vaccines costs and benefits of, 469–470 efficacy of, 464–466 Hib rapid assessment tool (Hib RAT), 467–468 hospital and community-based surveillance, 467 introduction, impediment to, 470 population-based surveillance, 467 immunization regimens, impact of in England and Wales, 454–455 in Netherlands, 455–456 in Republic of Ireland, 455 in United States, 454 incidence of, 452, 453 in England and Wales, 454–455 in Ireland, 455 serotype replacement disease, 469 Haemophilus influenzae type b (Hib) vaccines costs and benefits of, 469–470 efficacy of, 464–466 immunology and development, 453–454 introduction, impediment to, 470 licensure, 220 programmatic infant immunization, impact of The Gambia, 468 herd protection effect, 469 Kenya, 468–469 serotype replacement disease, 469 Haemophilus protein D, 219 HAI. See Serum hemagglutination inhibition (HAI) test Hantaan vaccine, 344 Hanta viruses, 284, 905, 907 hantavirus disease, 905 worldwide distribution of, 907 old and new world hantaviruses, 905, 906 transmission of, 905 vaccines for DNA vaccines, 910–911 HFRS- and HPS-associated hantaviruses, 908–909 inactivated virus vaccine, 905, 907–910 live-virus vaccine, 910 recombinant protein–based vaccines, 910 virus-like particles (VLPs), 910 HBsAg. See Hepatitis B surface antigen HBV. See Hepatitis B virus (HBV) HB vaccine, adverse effects, 245–246 HCMV. See Human cytomegalovirus (HCMV) infection HCV. See Hepatitis C virus HCV C–gpE1–gpE2 gene cassette, 614 HCV DNA vaccines, 609 advantages of, 612–613 disadvantages of, 613 immunogenicity in humans, 613 HCV infection, 603 natural immunity and immune correlates anti-E2 HVR1, 609 antienvelope antibody, 609 in chimpanzee challenge model, 607 HCV-specific T-cell responses, 607–609 human liver-chimeric mouse model, 609

[HCV infection] OCLN, CD81, SR-BI, CLDN1 expression, 606 HCV vaccine, efficacious potential benefit of, 603 with recombinant gpE1/gpE2 heterodimer, 610–612 using adjuvanted recombinant polypeptide subunits, 609 using disabled viral vectors, 614 Health Insurance Portability and Accountability Act (HIPAA), 28 Health level 7, 119 Health maintenance organizations, 247 Heat-labile enterotoxin (LT), 407 Heat-labile toxin, 714 Helicobacter pylori, 18, 81, 930 Helminth infections, 830 Hemagglutinin (HA), 533 Hematopoietic stem cells, 237 Hemorrhagic fever with renal syndrome (HFRS), 905, 908–909 Hendra (HeV) and Nipah virus (NiV), 885 active vaccines, 890 live recombinant vaccines, 890–891 subunit vaccines, 891–892 animal models, to study infection, 886–888 animal reservoirs for, 885 cellular tropism and host range of, 886 glycoproteins, 886 history of emergence of, 885 and human pathology, 885–886 passive vaccines antibodies, 889–890 fusion inhibitors, 889 therapeutic modalities for, 888 viral envelope glycoproteins and, 888–889 Hepatitis A, 236 Hepatitis B, 308, 322, 386, 390 core antigen, 309 vaccine, 42, 96, 102, 327 Hepatitis B surface antigen (HBsAg), 12, 264, 299 expression in tobacco plants, 307–308 vaccina recombinant, 342 Hepatitis B virus (HBV), 12, 81, 105 in hepatocellular carcinoma, 81 vaccine, 264, 267, 299–300 Hepatitis C, 239 Hepatitis C virus (HCV), 284, 293, 309, 364 cellular receptors interaction with of host lipoprotein, 606 genetic variability of, 604–605 genome, 603–604 infection with. See HCV infection models to study chimpanzee and mice, 605 retrovirus, 605–606 morphogenesis, 607 polyprotein, 604, 605 prevalence and genotype distribution, 604 replicons, 605 RNA replication, 603–604, 605 RNA translations, 603–604 transmission, 603 viral nucleocapsid protein core and the envelope glycoproteins, 604 virions, 606 HEPLISAVTM vaccine, 267 Herd immunity, 580 Herd protection effect, 469 Herpes simplex type-1 virus (HSVi), 419 Herpes simplex virus (HSV), 341–342, 352, 364, 390

999

Herpes simplex virus (HSV) infections, 653 herpesvirus family, 654 glycoproteins in, 654–655 replication of, 654 structure, 654 history of, 653–654 host immune responses correlation of disease with, 658 persistence of, 657–658 primary HSV infection and, 656–657 to recurrent HSV infection, 657 morbidity and mortality rate, 658 pathogenesis of, 655 animal models for study of, 655–656 exogenous reinfection, 655 first-episode infection, 655 transmission of infection, 655 vaccines for, 658–659. See also HSV vaccine, development of Herpesvirus Kaposi’s sarcoma herpesvirus (KSHV) , 930 Heterogeneous nuclear ribonucleoprotein-A1, 243 Heterologous immunity, 200 Heteropolymer-induced protection, 678–679 HFRS. See Hemorrhagic fever with renal syndrome (HFRS) Hhntavirus pulmonary syndrome (HPS), 905, 908–909 Hib. See Haemophilus influenzae type b Hib rapid assessment tool (Hib RAT), 467–468 Hib RAT. See Hib rapid assessment tool (Hib RAT) Highly active antiretroviral therapy (HAART), 634 Highly indebted poor countries, (HIPC), 98 Highly pathogenic avian influenza viruses (HPAIV), 545 HIPAA. See Health Insurance Portability and Accountability Act (HIPAA) HIPC. See Highly indebted poor countries (HIPC) Histidine triad proteins functions of, 484 HIV, 239, 284, 290, 293, 309, 322, 352–356, 365, 367, 386–387, 390, 790–791, 793–794, 830–831 DNA prime and NYVAC boost with HIV gag, pol, nef,and env immunogens, 401 DNA prime-MVA boost, 400 protective efficacy of HIV-1. See Human immunodeficiency virus type 1 (HIV-1) HIV vaccine, 289–291 biological challenges in development of, 585–586 clinical evaluation of glycoprotein antigens, envelope, 588 recombinant poxvirus vectors, 588–589 replication-defective viral gene delivery vehicles, 589 desirable characteristics of, 585 prospects for, 586–587 HL. See Hodgkin’s lymphoma HL-7. See Health Level 7 HLA-DR molecules, 277 HMOs. See Health maintenance organizations H1N1 virus, 547 H5N1 influenza vaccine, 255 HnRNP-A1. See Heterogeneous nuclear ribonucleoprotein-A1 H5N1 virus, 363 Hodgkin’s lymphoma vaccine formulation for, 646, 649 Holotoxins, 274, 544 Hookworm anemia, 831

1000

Index

Hookworm infection antigens under development, 833 disease burden, 831 epidemiology, 830–831 geographic distribution, 831 immunology, 831–832 vaccination by targeting adult blood feeding through specific antigens, 832–833 larval antigens, 832 vaccine. See Human hookworm vaccine (HHV) HPAIV. See Highly pathogenic avian influenza viruses (HPAIV) HPIV2 vaccine, live attenuated. See Live attenuated HPIV2 vaccine HPIV1 virus vaccines, live attenuated. See Live attenuated HPIV1 virus vaccines HPIV3 virus vaccines, live attenuated. See Live attenuated HPIV3 virus vaccines HPS. See Hantavirus pulmonary syndrome (HPS) HSCs. See Hematopoietic stem cells HSVi. See Herpes simplex type-1 virus (HSVi) HSV vaccine, development of, 659 genetically engineered HSV, for gliomas treatment, 662 animal models studies, 662–663 clinical experience, 662–663 follow-up investigations, 663 HSV DNA vaccines, 662 killed virus vaccines, 659–660 live vaccines, 661 antigens expressed in non-HSV vector, 661 genetically engineered viruses, 661–662 heterologous herpesviruses, 661 HSV mutants, 661 subunit vaccines, 660–661 wild-type virus vaccines, 659 HTLV-1. See Human T-lymphotropic virus type 1 Human cytomegalovirus (HCMV), 308 Human cytomegalovirus (HCMV) infection, 633 animal models of CMV, 635 guinea pig model, 635, 636 murine CMV models, 635 rhesus model, 635 as congenital disease, 633–634 cytomegalovirus, structure and replication of, 634 diseases caused by, 633 DNA vaccines for, 640–641 in HIV-infected individuals, 634 in immunocompromised patients, 634 live attenuated vaccines recombinant Towne/Toledo, 639 Towne vaccine, 638–639 prime boost strategies, 640 protection from, 634 cell-mediated immunity, role of, 635 innate immunity, role for, 634 role of antibody, 635 seroprevalence rates of, 633 subunit vaccines, 636 canarypox recombinants, 637 peptide vaccines, 637 recombinant adenovirus virus, 637 recombinant glycoprotein B vaccine, 636–637 replicon vaccines, 638 vaccinia, 637–638 vectored subunit vaccines, 637 vaccine for, development of, 635–636, 641 Human HLA transgenic mice, 648

Human hookworm vaccine (HHV), 830, 832–833 adjuvant technology, 834 adult hookworm antigens, 834 bivalent, 833 clinical development, 834–835 design and development, 835 development, 833–836 global access, 835 intellectual property and knowledge dissemination, 835 introduction and financing, 835 L3 hookworm antigens, 833–834 technology transfer, 834 Human immunodeficiency virus type 1 (HIV-1), 585 Human metapneumovirus, 363 Human papillomaviruses (HPV) vaccines, 122), 292–293, 300, 306, 319, 954 adverse events, after vaccination, 958 antibodies as marker for protection, 957–958 barriers to, use of, 958–959 deployment of, 958 disease caused by viruses, 954 and future developments, 959 immunotherapeutic vaccines, development of, 959 papillomavirus capsids natural immunity to, 954–955 production of, 955 structure and division of virus, 954 studies on safety and efficacy of, 955–957 Phase III studies, 955–956 Phase II studies, 955 Phase I studies, 955 technologies for, 955 Human parainfluenza, 366 Human pharmaceuticals, plant-derived, 311 Human T-lymphotropic virus type 1, 243 Humoral immunity, 580 Hyaluronidase, 484 IAP. See Intrapartum antibiotic prophylaxis (IAP) ICC-1132, 750, 751 Idiopathic thrombocytopenia, 245 IDNPE. See Immunodominant non-protective epitopes (IDNPEs) IFAT. See Indirect immunofluorescence antibody test (IFAT) IFN-g responses in infants, 230 IgA antibody, 273 IgA knockout mice, 534 IgE antibody, 253 IgG anti-PRP in young infants, 453–454 Immune deficiency, 264 Immune refocusing technology, 201–205 Immune response modifiers (IRMs), 846 Immune responses by antigen expression, 378 Immune system, 234 Immunization mucosal immunization aerosols, vaccines delivered as small-particle, 408–409 duration of protection, 406 immune effector responses, 406 inductive sites for immune responses, 405–406 nasal vaccines, 407 oral vaccines, 406 transcutaneous immunization (TCI) adjuvants, 417 bacterial diseases, applications for, 418–419 clinical product development, 421

[Immunization transcutaneous immunization (TCI)] Iomai dry patch vaccines, 422 methods for skin disruption miscellaneous diseases, applications for, 420–421 skin structure and immune function, 415–416 viral diseases, applications for, 419–420 Immunization, global. See also Expanded program on immunization (EPI) financial support to improve, types of, 102 Immunization schedules combination vaccines acellular pertussis vaccine, based, 437–438 regulatory aspects of, 436–437 safety evaluation of, 437 epidemiology of diseases and age of immunization Haemophilus influenzae type b, 433 neonatal tetanus, 433 Pertussis, 433 epidemiology of diseases and age of immunization Immunization schedules Streptococcus pneumoniae, 433 heterogeneity of DTP immunization schedules, 431–432 reasons, 430–431 immunological development, 434 numbers of doses and schedules comparing of, 434–435 proposed schedule, 438–439 vaccine incorporation, new, 438 Immunization strategies, 379–380 Immunization strategies, heterologous prime-boost antigen delivery systems and route, 396–397 anti-vector immunity, 397–398 clinical trials of cancer, 401–402 HIV, 400–401 malaria, 398–399 tuberculosis, 399–400 mechanisms, potential, 397 Immunodominance, 193 of B-cell determinants, 197–198 establisment of, 198 Leishmania model, 199 postnatal antigen exposure period, 199–200 postnatal bacterial colonization period, 198–199 prenatal germ line period, 198 nature of, 196 of T cell determinants, 193–197 Immunodominant non-protective epitopes (IDNPEs), 201 Immunology, 191 Immunopotentiating reconstituted influenza virosomes (IRIV), 290. See also Virosomes Immunoreceptor tyrosine kinase-based inhibitory motif (ITIM) , 931 Immunosenescence, 237, 240 Immunostimulating complexes (ISCOMs), 239, 255, 278, 292, 543, 750 adjuvants, 292 Immuno-suppressive therapy, 235 Improved disease models, 119 Improved vaccine adjuvants alternative approaches to, 284–286 needs for, 284–285 options for, 293 Improving manufacturing capabilities, 118 Inactivated influenza vaccines, 534–537

Index Inactivated ETEC vaccines inactivated whole cells in, 717–718 purified CFs in, 717 toxiods CTB, LTB, CTB/LTB hybrid, 716–717 LT, 716, 717 ST, 717 Inactivated polio vaccine (OPV), 875 Inactivated poliovirus vaccine (IPV), 430 IND. See Investigational new drug; Investigational new drugs (IND) Indirect immunofluorescence antibody test (IFAT), 910 Infectious bronchitis virus, 344, 364 bursal disease virus, 344, 364 hematopoietic necrosis virus, 389 Infectious diseases aging as risk from, 239 in elderly persons, 234 experimental adjuvanted vaccines against, 254 Infectious mononucleosis incidence of, 646 Inflammaging, 239 Inflammasomes, 168 Influenza, 532 HA-vaccinia recombinant, 342 immune responses to, 532–533 antigenic drift, 533 antigenic shift, 533 cellular immunity, 534 mucosal antibody, 533–534 serum antibody, 533 mortality rates, 532 pandemic of. See Pandemic vaccine development risks of, 532 strategy for control of, 532 vaccine, 288–289 vaccines for. See Influenza virus vaccines virus, 322, 363–367, 390 Influenza-ISCOM vaccine, 255 Influenza M2e-antigen, 277 Influenza vaccine, 150, 234, 239 Influenza virus vaccines adjuvants for, 542 combination formulations, 543 cytokines and chemokines, 543 emulsions, 542, 543 toll-like receptors (TLRs) agonists, 542–543 alternative substrates, for vaccine production, 541 egg-grown virus, problems in, 541 mammalian cell culture, use of, 541 MDCK cell–grown vaccine, 541 recombinant DNA techniques, use of, 541 cold-adapted influenza vaccine (CAIV), 537–538 adults and, 540–541 attenuation of master donor viruses, genetic basis, 538 children, studies on, 539–540 cold adaptation, process of, 537–538 development of, 537 and elderly population, 541 immune responses, 539 live attenuated viruses, use of, 537 protective efficacy of, 539–541 safety of, 538–539 DNA vaccines, 543 high- or low-dose vaccine, 542 inactivated influenza vaccines, 534 children and, 536 efficacy and effectiveness, 536 elderly adults and, 536 and healthy adults, 536

[Influenza virus vaccines inactivated influenza vaccines] high-risk groups and, 536–537 and revaccination, 537 safety of, 534–535 serum antibody responses, 535–536 intradermal and transcutaneous immunization, 542 intranasal inactivated influenza vaccine, 544 NA-based vaccines, 541–542 need of improvement, 541 potential live viral vaccine, 544 vaccine immune response, strategies for enhancing, 543–544 virosomal vaccine, 543 Innate immunity, 166–169, 239 Integrins, 176–178 Interagency coordinating committees (ICCs), 96, 98, 103, 104 Interleukin (IL)-2, 237 International Conference on Harmonisation (ICH), 60, 119 Q9 guideline, 150 International Finance Facility for Immunization (IFFIm) entities of GAVI foundation, 104 GAVI fund affiliate board, 104 international finance facility for immunization board, 104 working of, 73 Intestinal amebiasis, 826 Intradermal immunization approach, 542 Intranasal immunization with affinitypurified recombinant SCPA protein, 674 Intranasal vaccination, 854–855 Intrapartum antibiotic prophylaxis (IAP), 690 Investigational new drugs (IND), 27, 29, 116, 118 Investigational new drugs (IND) application, components of institutional review boards, 28 investigators, 28 monitors, 28 record keeping and product accountability for clinical trials, 28 reporting adverse experiences, 28 sponsors, 27 In vitro tissue culture manufacturing process, 149 In vivo expression technologies (IVETs), 19 Iomai dry patch vaccines animal models, testing in LT immunostimulatory patch for pandemic and seasonal flu, 422–424 trivalent inactivated split-influenza patch, 422 whole influenza virus patch, 422 clinical studies in humans dry versus wet patch for LT, 424–425 immune responses to injected H5N1 vaccine, improved, 426 travelers’ diarrhea illness, prevention and reduction of, 425–426 IPV. See Inactivated poliovirus vaccine (IPV) IRM. See Immune response modifiers (IRMs) Iron deficiency anemia (IDA), 830, 835 ISCOM. See Immunostimulating complexes (ISCOMs) ISCOMsTM vaccine, 255 ITIM. See Immunoreceptor tyrosine kinase-based inhibitory motif (ITIM) ITP. See Idiopathic thrombocytopenia Japanese encephalitis (JE) virus, 236 chimerivax vaccines ChimeriVax-JE structure and replication of, flavivirus, 557–559

1001

JCVI. See Joint Committee on Vaccination and Immunisation (JCVI) JE virus. See Japanese Encephalitis (JE) virus Joint Committee on Vaccination and Immunisation (JCVI), 431 Kala-azar. See Visceral leishmaniasis (VL) Kaposi’s sarcoma, 930 KFD. See Kyasanur forest disease (KFD) Killed parasite without BCG vaccine. See Mayrink’s vaccine Killed WC-rCTB Vaccine clinical trial of, 509 herd protection effect, 510 Killed whole-cell vaccines, for plague, 873 Knockout parasites, 794–795 KSHV. See Herpesvirus Kaposi’s sarcoma herpesvirus (KSHV) Kunjin virus, 364 Kyasanur forest disease (KFD) , 557 Lactobacillus casei, 13 Lactococcus lactis, 13 as vaccine vector, 683 Lactoferrin, 483 LAMP. See Lysosome associated membrane protein (LAMP) Langerhans cells (LCs), 415 Langerin (CD207), 334 Laryngotracheobronchitis, 620 Lassa fever, 363 Lassa virus, 365–366, 895 animal models, for study of infection, 896, 900 arenavirus family of, 895 phylogeny of, 896 chemokine, role in protection, 896–897 efficacy of Lassa candidate vaccines, 900–901 host for, 895 nonreplicating vaccines for alphavirus vector vaccine, 898 DNA vaccines, 898 inactivated virus particles, 898 peptide-based vaccination, 898 outbreaks of, 895 postexposure vaccination, 901 and replicating vaccines, 898 cell-mediated immunity, role of, 899 early studies on, 898–899 epitopes, identification of, 899 ML29 vaccine, 899–900 structure and genome, 897 transmission and prevalence, 895–896 vaccine for, 897–898 LBVs. See Live attenuated bacterial vaccines (LBVs) LCMV. See Lymphocytic choriomeningitis virus (LCMV) LCs. See Langerhans cells (LCs) Leishmania. See also Leishmaniasis vaccines against, 790–800 Leishmania amazonensis vaccine, 795–796, 798–799 Leishmania braziliensis, 799 Leishmania chagasi, 799 Leishmania donovani infection, 790, 792, 794, 796, 798–799 Leishmania guyanensis, 799 Leishmania infantum infection, 790, 792, 798–799 Leishmania major, 175 infection, 791–796, 798–799 Leishmania mexicana infection, 790, 792, 795–796, 799 Leishmania panamensis, 795

1002

Index

Leishmaniasis, 831, 836 disability-adjusted life years (DALYs), 790–791 form of, 790, 798 immunology, 791–792 vaccines ALMþBCG, 796 animal models for studies, 792–793 antigens for, 793 anti-Leishmania subunit vaccine, 796, 798 experimental, 793–794 first generation vaccines, 795–796 killed Leishmania major þ BCG, 796 killed Leishmania with and without adjuvants, 795–796 with Leishmania elongation and initiation factor, 799 leishmanization to evaluate, 793–794 live leishmania vaccines, 794–795 with LmSTI1 antigens, 799 Mayrink’s vaccine, 795 with recombinant antigens, 799–800 with recombinant protein vaccines, 796, 798 second generation vaccines, 796–798 therapeutic vaccine, 799–800 with thiol-specific-antioxidant antigens, 799 in use, 793 Leishmanin skin test (LST), 795–796 Lethal factor, 851 LF. See Lethal factor Lipid core peptide (LCP) system, 318–319 Lipopeptide-based vaccines, 315–323 action mechanism, 321–322 advantages, 316 delivery and adjuvanting effects, 320 epitope identification, 317–318 limitations, 316–317 structures, 318–320 Lipopeptide with antigen, codelivery of, 322 Lipopolysaccharide (LPS), 286, 293, 322, 330 Lipoproteins (LPPs), 286 PiuA and PiaA, 484 Lipoteichoic acid (LTA), oral administration of, 680 Liquid formulation of Ty21a, 498 Listeria monocytogenes, 13, 175 Live anthrax vaccines development of, 855–856 live attenuated recombinant Bacillus anthracis MASC-13, 856 Salmonella vectors, 856 STI-1 and A16R, 855 Live attenuated bacterial vaccines (LBVs), 13 Live attenuated ETEC candidate vaccines attenuated Vibrio cholerae and Salmonella strains, 719 expressing CFs, 719 immune response in children, 720 multivalent Shigella/ETEC vaccines, 719 potential of, 718 Live attenuated HPIV2 vaccine, 629 Live attenuated HPIV1 virus vaccines development strategies, 628 non-chimeric HPIV1 vaccine, 628–629 Live attenuated HPIV3 virus vaccines B/HPIV3 and HPIV3-PB, 628 HPIV3cp45, 627–628 Live attenuated measles vaccines aerosol administration of, 580 limitation, 579 Live attenuated PIV vaccine, 623 development principles, 624–625

Live attenuated RSV vaccines, 623 bovine RSV, 626–627 development principles, 624–625 RSVcp248/404/1030DSH deletion of NS1, NS2, and M2-2, 626 mutant virus of, 625 against subgroup B, 627 Live attenuated spore vaccines, 852–853 Live-attenuated vaccines, for plague, 873 Live attenuated virus (LAV) vaccines, for dengue, 594, 595 candidates in clinical trials, 596 Acambis ChimeriVax vaccines, 597 chimeric vaccine candidates, 596–597 DENV-2 PDK-53 vaccine, 597 genetically engineered candidates, 596 LID/NIAID D30 vaccines, 597 Mahidol vaccine candidates, 596 primary dog kidney (PDK) cells, candidates grown in, 596 WRAIR candidates, 596 Live oral typhoid vaccine, 501 CVD 908, 499–500 CVD 909, 500 CVD 908-htrA, 500 future use of, 503 M01ZH09, 502 PBCC 211 and PBCC222, 500, 502 Ty21a. See Ty21a Ty445 and Ty800, 502 541Ty and 543Ty, 499 X3927, X4073, and X8110, 502 Lower respiratory infections, 831 Lower respiratory tract illness due to RSV and PIV, 620 LOX-1, 334–335 LRTI. See Lower respiratory tract illness L-selectin (CD62L), 238 LT. See Heat-labile enterotoxin (LT); Heat-labile toxin LT holotoxins, 275 LT induce expression, 278 LT(R192G) mutant, 275 LuminexTM, 222 Luteinizing hormone releasing hormone (LHRH), 317 Lutzomyia, 791 Lyme disease, 363 Lymphatic filariasis, 831 Lymphocyte trafficking, 178 Lymphocytic choriomeningitis virus (LCMV), 895 Lymphopoiesis, age-associated alterations, 237 Lysosome associated membrane protein (LAMP) , 942 MAbs. See Monoclonal antibodies (MAbs) Macrophage-activating lipopeptide-2 (MALP-2), 320 Macrophage scavenger receptor, 167 Madin-Darby canine kidney (MDCK) cells, 541 Major histocompatibility complex, 239 Malaria, 284, 386, 390, 793, 830–832 in endemic areas, 746 Plasmodium falciparum, as causative organism, 746 life cycle of, 746–748 ‘‘two-tiered’’ vaccine defense, 748 vaccine for, 746 adjuvants, use of, 750–751 blood-stage vaccine. See Blood-stage malaria vaccines pre-erythrocytic malaria vaccines. See Pre-erythrocytic malaria vaccines and public health objectives, 748–749 recombinant protein and peptide subunit vaccines, 749–750

[Malaria vaccine for] roadmap for vaccine development, 762 and strategic approaches, 762–763 targeting parasite life cycle, 748 transmission-blocking malaria vaccines. See Transmission-blocking malaria vaccines (TBMV) Malaria, vaccine trials protective efficacy of DNA prime-MVAboost, 399 FP9 ME-TRAP prime-MVA ME-TRAP, 399 Malaria vaccines, gene-based clinical studies adenovirus vectors, 778 DNA vectors, 776 DNA vectors in prime-boost regimens, 777 poxvirus vectors, 777 preclinical studies adenovirus vectors, 776 DNA vectors, 775 heterologous prime-boost regimens, 775–776 prospects of, 778–710 Mannheimia haemolytica, 308 Mannose receptor (CD206), 334 MAPKK. See Mitogen-activated protein kinase kinase Marburg virus, 352, 363, 366 Master cell bank, 147 Mastomys natalensis, 895 Maternal antibodies influence on early-life antibody responses, 230 on neonatal CD4þ and CD8þ vaccine responses, 231 maternal IgG antibodies (MatAb), 230 Mayrink’s vaccine, 795 MCB. See Master cell bank MCC vaccine. See Meningococcal C vaccine MDC. See Myeloid-derived dendritic cells MDCK. See Madin-Darby canine kidney (MDCK) cells MDP. See Muramyl dipeptide Measles antibodies, avidity of, 580 approaches for protecting young infants against, 579–580 in developing countries, 579 immunological correlates of protection against, 580 immunosuppression in, 580 Measles DNA vaccine DNA vectors, 582 prime-boost approach of, 580–581 Rhesus macaques immunization with, 580 Sindbis replicon-based measles DNA vaccine biodistribution studies, 582 development, 581 efficacy studies, 581–582 immunogenicity studies, 581 integration studies, 582 phase I trial, 582 toxicology studies, 582 Measles elimination program, 580 Measles-mumps-rubella vaccination, 245 Measles vaccine desirable characteristics for, 580 Measles virus (MV), 308, 352–353, 364–366, 390, 831 transmissibility, 579 Mebendazole, 830 MedImmune, 639 Melanoma, 389 antigens, 364

Index Memory T-cell differentiation, models, 176, 177 Mengo virus, 364 Meningococcal capsular polysaccharides, 456 Meningococcal C disease in England and Wales, 457 epidemiology of, 453 pathophysiology of, 456 Meningococcal C vaccine capsule switching, 458 correlates of protection in, 456 immunization impact in Australia, 459 in Canada, 458–459 effectiveness estimates, 457 in England and Wales, 456–457 in European Union countries, 458 in Republic of Ireland, 458 in United States, 459 immunology and development, 456 T cell–dependent antigens, 456 Meningococcal disease. See Meningococcal vaccines, conjugate and protein-based Meningococcal outer-membrane protein (OMP), 219 Meningococcal Reference Unit, 458 Meningococcal vaccines, conjugate and protein-based commensal neisseriae and attenuated bacteria, 448 genome-based discovery of new antigens, 447–448 lipopolysaccharide (LPS) vaccines, 446 outer membrane protein vaccines (OMPs) outer membrane vesicle (OMV) vaccines, 445 PorA proteins serogroups of meningococci, 443 Meningococcus, 452 Mesocricetus auratus, 831 Meso Scale Discovery (MSDTM) technology, 222 Metapneumovirus, 13 Methodological approaches for safety and protection of vaccine direct approaches case-control studies, 53 controlled, cohort studies, 53 variant designs, 54 indirect approaches, 53 methodological limitations of observational vaccine evaluations, 55 population-based databases, increasing importance of Vaccine Adverse Event Reporting System (VAERS), 54 population-based databases, increasing importance of, 54 ME-TRAP. See Multiple epitopethrombospondin-related adhesionprotein (ME-TRAP) MF59 formulation, 253 MHC. See Major histocompatibility complex Microbial contamination, recommended limits, 148 Microparticles as vaccine adjuvants, 290 Miltefosine, 791 Mink enteritis virus, 309 Mitogen-activated protein kinase kinase, 851 MMR-related ITP, 245 MMR vaccination. See Measlesmumps-rubella vaccination MNC partners, 152 Modified vaccinia Ankara (MVA), 15, 396, 398, 522, 775, 839 virus, 340, 355 Monkeypox virus, 340 Monoclonal antibodies (MAbs), 914, 917–918

Monophosphoryl lipid A (MPL), 265, 274, 286, 293, 322 MontanideTM ISA 51 and ISA 720, 254 Montenegro skin test, 795 Morbilliviruses, 366 MPL. See Monophosphoryl lipid A M protein. See Streptococcal M protein MRU. See Meningococcal Reference Unit MS. See Multiple sclerosis MSC. See Myeloid suppressor cells (MSC) MSR. See Macrophage scavenger receptor MTB. See Mycobacterium tuberculosis MTB auxotrophs, 521 Mtb72f, 522 MTB72f vaccine, 526 MTB 30 kDa major secretory protein BCG as vector for delivery of, 523 expression by rBCG30, 523 MTP-PE. See Muramyl tripeptide dipalmitoyl phosphatidylethanolamine Mucosal adjuvant activity, 264 Mucosal antibody, 533–534 Mucosal immune responses, 716 Mucosal immunization, 273 Mucosal leishmaniasis (ML), 790 Mucosal vaccine for serotype-independent protection, 679 Multiepitope vaccines, 14 Multifunctional T Cells, 175 Multiple-antigen lipophilic adjuvant carrier (MALAC) system, 317, 319 Multiple epitope-thrombospondin-related adhesionprotein (ME-TRAP), 776, 777 Multiple sclerosis, 175, 242, 245–246 Multiplex assays for measuring specific antibodies, 222 Multiuse nozzle jet injectors (MUNJIs), 409–410 Multivalent M protein–based vaccines clinical trials of, 685–686 rationale for, 684 Multivalent Shigella/ETEC vaccines, 719 development of, 725–726 MUNJIs. See Multiuse nozzle jet injectors (MUNJIs) Muramyl dipeptide, 254, 274 Muramyl tripeptide dipalmitoyl phosphatidylethanolamine, 254–255 Murray valley encephalitis (MVE) , 557 Mutants of shigella, enteroinvasive deletion aromatic auxotrophs auxotrophic S. flexneri 2a vaccine candidates, 705 auxotrophic S. flexneri 2a with a deletion in virG, 705 auxotrophic S. flexneri g vaccine Candidates, 705 attenuated with a mutation in virG, 707–708 purine auxotrophs, 705, 707 Mutations, aro, 13 MV. See Measles virus MVA. See Modified vaccinia Ankara (MVA) MVA85A vaccine human studies, 526–527 immunogenicity and efficacy in animal models, 526 MVE. See Murray valley encephalitis (MVE) Mycobacterium bovis Bacille CalmetteGue´rin, 815 Mycobacterium leprae, 175 hsp60 protein, 522, 523 Mycobacterium phlei, 255 Mycobacterium tuberculosis, 8, 175, 234, 235, 265, 322, 387, 396, 400, 516, 732 attenuated mutants of MTB auxotrophs, 521 biology of, 517

1003

[Mycobacterium tuberculosis] immunology of CD4 T cells, 517–518 cytokine secretion pattern, 518 memory development, 519 phagosome maturation, 517 T-cell response, 517 T lymphocytes, 517 Mycobacterium vaccae heat-killed, 523 Mycolyl transferase, 523 Myeloid-derived dendritic cells, 263 Myeloid suppressor cells (MSC), M01ZH09 (live oral typhoid vaccine), 502 Na-ASP-2 hookworm vaccine, 833–834 NAI. See Naturally acquired immunity (NAI) NA inhibition (NI), 533 NALT. See Nasal-associated lymphoid tissue (NALT) Nasal-associated lymphoid tissue (NALT), 276, 405, 674 Nasal colonization, protection against, 485 Nasal vaccines accuspray nasal spray device, 407 clinical trials, 407–408 intranasal adjuvants, 408 Nasopharyngeal carcinoma prophylactic screening and posttherapy monitoring, 650 vaccine formulation for, 646, 647, 649, 651 National immunization days (NIDs), 96 National immunization program (NIP), dengue vaccine in, 598 National Institute of Allergy and Infectious Diseases (NIAID), 17, 589, 885 National Institute of Health, 122 National regulatory authorities (NRAs), 98, 121 assessments conducted against indicators, 124 prequalification process, 125–127 role in prequalification, 123 strengthening activities, 123, 129 Natural killer (NK) cells, 167, 539 Naturally acquired immunity (NAI), 749 Necator americanus, 830–833 Needle-free devices jet injectors LectraJet, 410 Mini-Imojet, 410 Negative strand RNA virus, 361, 365–367. See also RNA virus influenza virus, 366–367 non-segmented, 365–366 paramyxoviruses, 366 rhabdoviruses, 365–366 segmented, 366–367 Negative vaccination, 969 Neisseria flavescens, 448 Neisseria lactamica, 448 Neisseria meningitides, 13, 150, 284, 291, 448 serogroups B and C, 452 Neonatal tetanus (NT), 91, 93, 94, 95 NERVE. See New Enhanced Reverse Vaccinology Environment (NERVE) Neuraminidase (NA), 533 NanA and NanB, 484 Neuraminidase vaccines, 541–542 Neurofibrillary tangles, 962, 963 Newcastle disease, 344, 366 New Enhanced Reverse Vaccinology Environment (NERVE), 15 NF-kB. See Nuclear factor-kappaB NIAID. See National Institute of Allergy and Infectious Diseases (NIAID) Nicotine addiction, 303 NicVax, nicotine vaccines, 985–986

1004

Index

NIDs. See National immunization days (NIDs) NIH comparison trial of multiadjuvanted HIV gp 120 vaccine, 253 NK. See Natural killer (NK) cells NKT lymphocytes, 330 Nonantibiotic plasmid selection systems, 378–379 Non-Hodgkin’s lymphoma (NHL), 310–311 Nonionic block copolymers, 255–256 Non-propagating virus vectors from positive strand RNA virus, 362 Nonreplicating virus vaccines, 622–623 Non-segmented negative strand RNA virus, 365–366 Nontoxic LTK63 mutant, 277 Norovirus (NoV), 300, 309 Norwalk virus capsid protein (NVCP), 308 Norwalk virus (NV), 293, 300, 308, 363 NoV. See Norovirus (NoV) NPC. See Nasopharyngeal carcinoma NRA. See National regulatory authority NS3-4A protease complex, 604 NS3-4A serine protease complex, 604 NS4B and NS5B (integral membrane protein), 604 NT. See Neonatal tetanus (NT) Nuclear factor-kappaB, 274 Nucleotide-binding oligomerization domain (NOD) proteins, 167, 274 NV. See Norwalk virus (NV) NYVAC vaccinia virus, 344–345 OAU. See Organization of African Unity (OAU) ODN. See Oligodeoxynucleotides OIC. See Organization of Islamic Conferences (OIC) Oligodeoxynucleotides, 256, 267, 268 OMPs. See Outer membrane protein vaccines (OMPs) OMV. See Outer membrane vesicle (OMV) vaccines OPA. See Opsonophagocytosis assay Opsonophagocytosis assay, 221 OPV. See Oral poliovirus vaccine (OPV) Oral poliovirus vaccine (OPV), 132, 875 Oral Vaccines, 406 Organization of African Unity (OAU), 156 Organization of Islamic Conferences (OIC), 156 Orthopoxvirus ectromelia virus (ECTV), 846 Orthopoxviruses, 363. See also Smallpox vaccines, development of Osp A Lyme disease vaccine, 134–135 O-specific polysaccharides, 489 backbone structure, 492 immunogenicity, 493 O-SPs. See O-specific polysaccharides Outer membrane protein vaccines (OMPs), 444 Outer membrane vesicle (OMV) vaccines. See also Meningococcal vaccines, conjugate and protein-based bactericidal activity, 445 clinical trials of, 445–446 PA. See Protective antigen PA-based vaccines, 852 clinical trials of, 858 PAHO. See Pan-American Health Organization PAHO Executive Committee, 122 PAMPs. See Pathogen-associated molecular patterns Pan-American Health Organization (PAHO), 67, 119, 122

Pandemic vaccine development, 544 for avian H9N2 strains, 547 history of, 544–545 avian influenza A viruses, 545 whole-virus formulations, 544–545 for H2N2 viruses, 547 for H5N1 viruses, 545–546 adjuvanted H5N1 vaccines, 546–547 live attenuated virus vaccines, 547 subvirion H5N1 vaccines, 546 whole-virus H1N1 vaccines, 546 and licensing criteria, 547 stockpiling of pre-pandemic vaccine, 547–548 Pan-genome, 18–19 Papillomavirus, 365 Parainfluenza viruses, 352, 363–364 genomes of, 620 immunity to, 620–621 PIV1, PIV2, and PIV3, 620 Paramyxoviruses, 363, 366 Paratyphoid fevers incidence rates, 497 Paromomycin, 791 Passive surveillance systems, 137–138 Pasteurella septica, 3 Pasteur’s general principle, 3, 12 PAT. See Process analytical technology PATH. See Program for Appropriate Technology in Health Pathogen-associated molecular patterns (PAMPs), 167, 285–286, 293 Pathogenesis cholera disease, 507 shigella infection inflammatory destruction of the intestinal epithelium, 701–702 invasive phenotype, virulence plasmid of shigella, 701 role of toxin in, 702 Staphylococcus aureus infection, 728–730 Pathogens, classification of, 192–193 class II pathogens, 192–193 class I pathogens, 192 Pattern recognition receptors (PRRs), 167, 263, 274, 285–286 PBCC 211 and PBCC222 formulation, 500 PBMCs. See Peripheral blood mononuclear cells; Peripheral B memory cells PC. See Phosphocholine PcpA, leucine-rich protein, 485 PCV. See Pneumococcal conjugate vaccine (PCV) PDC. See Plasmacytoid dendritic cells PDPs. See Product development partnerships (PDPs) PDUFA. See Prescription Drug User Fee Act of 1992 PDUFA III. See Public Health Security and Bioterrorism Preparedness Act of 2002 PDVI. See Pediatric dengue vaccine initiative (PDVI) PE. See Protective efficacy (PE) Pediatric dengue vaccine initiative (PDVI), 599 Pelvic inflammatory disease (PID), 737 PEP. See Postexposure prophylaxis Peptide–based polymers, preparation of, 317–318 Peripheral blood mononuclear cells, 223 Peripheral B memory cells, 223 Peripheral T-cell pool, age-related changes, 238 Pertussis toxin, 12 PfEMP1. See P. falciparum erythrocyte membrane protein 1 (PfEMP1) Phage libraries, 890

Phlebotomus, 791 Phlebotomus papatasi, 793 Phosphocholine, 482 Picornaviruses, 361, 363–364 PID. See Pelvic inflammatory disease (PID) Pilus subunit, 485 PIV infection, 620 PIVs. See Parainfluenza viruses PIV vaccines desirable characteristics of, 622 obstacles in developing, 621–622 vectored, 629 Plague pathology, 872 vaccines characteristics, 874 killed whole-cell, 873 live-attenuated, 873 prime-boost vaccination, 874–875 subunit, 873–874 Yersinia pestis, 871–872 lifestyle, 872–873 virulence factors and protective antigens, 871–872 Plant-derived vaccines as alternative expression system, 309 cancer vaccines, 310–311 clinical trials, 307–308, 311 expression of antigenic proteins, 306–307 human pharmaceuticals, 311 issues and challenges, 311 multivalent and multicomponent, 308 plastid transformation, 310 production and delivery, 306 protection against pathogen, 308 targets, 310 Plants expressed antigens targets, 310 expression of antigenic proteins in, 306–307 production and delivery of vaccine in, 306 viral vectors as alternative expression system, 309 Plaque reduction neutralizing (PRN) antibodies, 407 Plasmacytoid dendritic cells, 263 Plasmid-based expression technologies engineering immune responses, 378 foreign antigen expression, 375–377 toxicity, 377–378 innovations in, 375–379 nonantibiotic plasmid selection systems, 378–379 plasmid stability optimization, 378 Plasmid DNA vaccines, 811, 856–857, 859 Plasmodium berghei, 774, 775 Plasmodium falciparum, 264, 302, 345, 364, 396, 398, 774, 775 asexual erythrocytic stage vaccines, 786–788 Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), 747–748 Plasmodium falciparum Sporozoite Vaccine, 784–786 Plasmodium knowlesi, 774, 775 Plasmodium yoelii, 251, 774, 775 Plastid transformation, 310 PLG-CpG ODN combination, 278 Plum poxvirus, 309 Pnc vaccine, 221 Pneumococcal common protein vaccine clinical considerations for, 485–486 Pneumococcal conjugate vaccine (PCV) clinical trials of carriage, 474 otitis media, 474 vaccine protection, duration of, 475 vaccine safety, 476

Index [Pneumococcal conjugate vaccine (PCV)] routine use and effects of carriage and herd immunity, 478–479 vaccine effectiveness, 476 vaccine schedules, 477–478 Pneumococcal disease and carriage epidemiology of pneumococcal vaccines clinical trials of, 474–476 routine use and effects of, 476–479 Pneumococcal infection protection against, 483 Pneumococcal polysaccharide vaccine, 489 Pneumococcal protein(s) autolysin, 484 as carriers for polysaccharides, 485 functions of, 482 in pathogenesis of pneumococcal disease, 485 PcsB, 485 pneumolysin antibodies to, 484 cytotoxicity, 483 functions of, 483 protection against nasal colonization, 485 protective efficacy in mice, 483 PsaA, 484 PspA antibody to, 483 binding with lactoferrin, 483 structural variability, 482 PspC, 484 Pneumococcal surface antigen A. See PsaA Pneumococcal surface protein A. See PspA Pneumococcus protection-eliciting molecules of, 483 Pneumolysin antibodies to, 484 cytotoxicity, 483 functions of, 483 Pneumonic plague, 871–874 Point mutations, 234 Polio eradication areas of highly efficient poliovirus transmission, 157 areas of suboptimal strategy implementation, 157–158 circulating vaccine-derived polioviruses, 159–160 criteria for costs and benefits, 154 societal and political considerations, 155 technical feasibility, 153–154 gaps in poliovirus surveillance, 159 history of, 155–156 OPV cessation risk reduction and management, 161–162 risks associated with, 160–161 reinfection of previously polio-free areas, 158 vaccine-associated paralytic poliomyelitis (VAPP) Poliomyelitis, eradication of. See Expanded program on immunization (EPI) Poliovirus, 353, 361, 364 Poly-g-D-glutamic acid capsule, 851 role in protection against lethal challenge, 854 Polylactide/polyglycolide (PLG) microspheres, 274 Poly-N-acetylglucosamine (PNAG), 730 Polyribosylribitol phosphate (PRP), 219, 464 efficacy in bacteremic Hib disease, 454 serum IgG antibodies to, 453–454 unconjugated, 454 Polysaccharide-protein conjugates, 485 vaccines, 452 Polysaccharide vaccines, 489 Porphyromonas gingivalis, 17

Positive strand RNA virus non-propagating virus vectors, 362 propagation-competent virus vectors, 361–362 replican vectors, 362–365 vaccine vectors from, 361–365. See also RNA virus Postexposure prophylaxis, 857 Postexposure vaccines efficacy, 522 Post-kala-azar dermal leishmaniasis (PKDL), 796 Posttransplant lymphoproliferative disease immunotherapy integration with treatment protocols, 650 incidence of, 646 PotD, surface transporter molecule, 484 Potyviruses, 309 Poxviruses development of expression vectors, 340–342 expression of foreign genes, 341 fidelity of expression, 341–342 hybrid expression vectors, 341 insertion of foreign DNA into poxvirus genome, 340–341 isolation procedures, 341 Praziquantel (PZQ), 808, 810, 815 Pre-erythrocytic malaria vaccines, 751, 753 in clinical development, 752 circumsporozoite protein (CSP), 752 liver-stage antigens, 753 thrombospondin-related adhesive protein (TRAP), 752–753 development of, 752 Prescription Drug User Fee Act of 1992, 116 Primary EBV infection. See EBV infection, primary Primary prophylaxis of streptococcal pharyngitis and rheumatic heart disease, 673 Prime/boost settings adjuvant MF59 use in, 289–290 Prime-boost vaccination for plague, 874–875 PRN. See Plaque reduction neutralizing (PRN) antibodies Probabilistic age pyramid, 235 Process analytical technology (PAT), 150 launched by FDA, 150 Product development partnerships (PDPs), 73 Products regulation, manufactured for use outside the country, 128 Program for Appropriate Technology in Health, 128 Propagation-competent virus vectors, 361–363 Prophylactic infectious disease vaccines, 265 Prophylactic vaccine and primary EBV infection, 646 Protective antibodies, 170–171 Protective antigen antibodies to, 851 crystal structure of, 851 expression in Salmonella typhimurium, 856 Protective efficacy (PE), 42 Protein array technology, 20 Protein-conjugated Haemophilus influenzae type b (Hib) vaccines Protein vaccines receptors for targeting, 334–335 targeting to maturing DCs, 330–333 Proteomics, 20 Protocol development for clinical investigation of a new candidate vaccine components of, 29 studies involving children and infants, considerations for, 29 vaccines prepared by recombinant DNA technology, considerations for, 30 vaccines that can be transmitted person-toperson, considerations for, 30

1005

Protollin, 920 PRP. See Polyribosylribitol phosphate (PRP) PRRs. See Pattern recognition receptors PsaA, 484 Psammomys, 792 Pseudomonas aeruginosa, 309 PspA antibody to, 483 binding with lactoferrin, 483 structural variability, 482 PspC domain structure of, 484 role in adherence and colonization, 484 PTLD. See Posttransplant lymphoproliferative disease Public Health Security and Bioterrorism Preparedness Act of 2002, 116 Public Health Service (PHS) Act, 116 of 1944, 116, 122 QS-21 (StimulonTM), 255 Quality assurance, 147 RA. See Rheumatoid arthritis (RA) Rabbit hemorrhagic disease virus, 308–309, 420 Rabies, 236 Rabies glycoprotein-vaccinia recombinants, 342, 344 Rabies virus (RV), 308–309, 344, 352–353, 361 Randomized clinical trials (phase III) of vaccines, additional issues for analyzing data, issues in, 51–52 compared agents, issues in allocation of, blocked allocation, 47 stratified allocation, 47 compared agents, issues in the administration of, 48 demarcating the type of vaccine protection to be measured, 44 Good Clinical Practice (GCP), 52 perspective of the trial, clarification of, 44–46 research questions for the trial, 43–44 sample size estimation and interpretation of background information, 46–47 surveillance, issue in detection bias, 50–51 scope, 49 time frame, 50 RBCG30 (recombinant BCG vaccine) human studies, 524 immunogenicity and efficacy in animal models, 523–524 MTB 30 kDa protein expression by, 523 rBCG(mbtB)30, 524 RBCG strain expressing cytolysin human studies, 524–525 immunogenicity and efficacy in animal models, 524 rationale for constructing, 524 RBCG vaccine escaping phagosome, 521 expressing MTB proteins, 521 secreting cytokines, 521 RB/HPIV3 chimeric virus, 629 RCTB-CF ETEC vaccines, 718 Reaching every district (RED) strategy Components of, 97 Reactive nitrogen species (RNS), 936 Reactive oxygen species (ROS), 936 Recombinant adenovirus (rAd) vectors. See also Adenovirus recombinants vaccines, 353–355 Recombinant AD5 (rAD5) vaccines vector, 353–355

1006

Index

Recombinant DNA technology conjugate vaccines, 13 DNA vaccines, 14–15 engineering live attenuated vaccines, 13 live attenuated bacteria as vectors, 13 replicating and nonreplicating vectors, 14 virus-like particles (VLPs), 14 Recombinant HCV protein vaccines, 610–612 Recombinant PA–based vaccines aluminum-based adjuvants, 853 clinical trials of, 858–859 PA combined with adjuvants, 853–854 PA interaction with LF and EF, 854 protection against aerosol challenge, 853 T-independent antigen, 854 Recombinant poxvirus isolation procedures, 341 Recombinant Streptococcus gordonii, vaccine trials with, 683–684 Recombinant subunit vaccines, 14 Recombinant vaccinia virus preparation, 340–341 vaccines clinical applications, 344–345 experimental, 342 immunogenicity, 342–343 safety, 343–344 veterinary applications, 344 Recombinant viral vectors, 856 RED. See Reaching every district (RED) strategy Regulation, of vaccines, 121 Regulatory issues in clinical trials of new vaccine candidate investigational new drugs application, components of, 27 regulation of vaccine development, history of, 27 regulatory considerations in international trials, 29 Regulatory T cells, 175, 244 Replacement live mycobacterial vaccines, 521–522 Replican vectors advantages and disadvantages, 364–365 alphaviruses, 362–364 coronaviruses, 364 flaviviruses, 364 picornaviruses, 364 from positive strand RNA virus, 362–365 Replication-competent Ad-recombinant vaccines, 353–355 Replication-defective Ad-recombinant vaccines, 355–356 Replicon vaccines, from RNA virus, 361–365 Respiratory syncytial virus (RSV), 308–309, 352, 363, 365, 419 electron micrograph of, 621 genomes of, 620 immunity to, 620–621 infection, 620 vaccines clinical trials of, 623 desirable characteristics of, 622 live attenuated. See Live attenuated RSV vaccines nonreplicating, 622–623 obstacles in developing, 621–622 vectored, 629 vaccinia recombinants, 342 Rhabdoviruses, 363, 365–366 Rhesus macaques immunized with measles DNA vaccine, 580 measles antibodies in, 580 Rheumatic heart disease antibiotic prevention of, 673 prevalence of, 672

Rheumatoid arthritis (RA), 175, 975 Rhinoviruses, 364 Ribavirin therapy, 888 Rinderpest viruses, 366 Risk management standards, 150 Rituximab, 650 RNA virus replicon vaccines, 361–365 vaccines vectors from negative strand, 365–367 non-propagating virus vectors, 362 nonsegmented negative strand, 365–366 from positive strand, 361–365 propagation competent vectors, 361–362 replicon vectors, 362–365 segmented negative strand, 366–367 vectors, 363 RNS. See Reactive nitrogen species (RNS) ROS. See Reactive oxygen species (ROS) RotarixTM vaccine, 573, 575 RotashieldTM vaccine, 572–574 RotaTeqTM vaccine, 572, 573, 576 Rotavirus, 308–309, 352–353 Rotavirus vaccine future considerations, 576 importance of, 570 mechanisms of protection, after live virus infection, 573 NA not only effector of protection, studies on, 574–575 role for neutralizing antibody (NA) in, 573–574 mortality rate, rotavirus disease and, 570, 571 nonliving vaccine candidates, 575–576 inactivated rotavirus particles, 575 rotavirus VP6 protein, 575–576 virus-like particles (VLPs) vaccines, 575 rotavirus natural infection, protective effect of, 571 replication cycle of, 570–571 structure of, 570, 572 vaccine candidates, development of, 573 animal rotavirus strains, 572 from infected humans, 572 monovalent candidates, 575 multivalent reassortant vaccines, 574 neonatal strains, 573 Rotavirus Vaccine Project of PATH, 129 RPA vaccines, 858–859 RSV. See Respiratory syncytial virus (RSV) RTS,S vaccine, 746, 750–753 Rubella, 353 S. boydii, 700, 703 SAARC. See South Asian Association For Regional Cooperation (SAARC) Sabin and Salk polio vaccines, 327 Saccharomyces cerevisiae, 795 SAF. See Syntex adjuvant formulation Salmonella, 815 Salmonella dublin, 825 Salmonella enterica, 13, 26 Salmonella enterica serovar Typhimurium (S. Typhimurium), based live vectors. See Salmonella enterica serovar Typhi (S. Typhi) Salmonella enterica serovar Typhi (S. Typhi), 375, 825 chromosomal antigen expression strategies, 379 DNA vaccines delivery, 380–382 immunization strategies, 379–380 as live vectors, 375–382, 874 plasmid-based expression technologies and, 375–379 engineering immune responses, 378

[Salmonella enterica serovar Typhi (S. Typhi) plasmid-based expression technologies and] genetic optimization of plasmid stability, 378 nonantibiotic plasmid selection systems, 378–379 reduction of antigen toxicity, 377–378 regulated expression of foreign antigens, 376–377 Salmonella minnesota, 799 Salmonella outbreaks, causes of, 492 Salmonella paratyphi A LPS serum antibodies elicited by, 493 O-acetyl groups, 493 vaccines for, 492–493 Salmonella paratyphi A strains CVD 1902 and CVD 1903, 502–503 multiresistant, 497 Salmonella pathogenicity island 2, 502 Salmonella typhi attenuated strains of Ty2. See Ty2 auxotrophic mutants of, 499 multiresistant strains of antibiotics against, 497 Ty21a. See Ty21a Ty445 and Ty800, 502 541Ty and 543Ty, 499 Salmonella typhimurium conjugate vaccine, 493 Salmonella typhimurium strain deletions in cya and crp, 502 Salmonella typhi-Shigella sonnei (FS) hybrid vaccine, 703–704 vaccine, 703–704 SALT. See Skin-associated lymphoid tissue (SALT) Saponins, 239, 255 SARS. See Severe acute respiratory syndrome (SARS) SAVINE. See Scrambled antigen vaccine SBA. See Serum bactericidal activity (SBA) SC. See Stratum corneum (SC) Schistosoma, 808–815 Schistosoma haematobium, 808–810, 814 human immunity to, 810 life cycle, 809 Schistosoma japonicum, 808–815 human immunity to, 810 life cycle, 809 Schistosoma mansoni, 808–815 human immunity to, 810 life cycle, 809 Schistosomes, immune response to, 809–810 Schistosomiasis, 831, 836 characteristics, 808 humans and resistance to, 810 immune response to schistosomes and, 809–810 vaccines, 808–815 antigens, 814–815 calpain, 811, 813 candidates, 811–814 cytosolic Cu–Zn superoxide dismutase, 811 fatty acid binding protein, 811, 813–814 ferritin, 813 glutathione-S-transferase, 811–812 paramyosin, 811, 813–814 recombinant vaccines, 811 S. japonicum vaccine antigens, 813 S. mansoni vaccine antigens, 812 serpin, 813 Sm29 and other new membrane proteins, 813 superoxide dismutase, 811, 813–814 tetraspanins, 812–813 triose phosphate isomerase, 811, 813

Index SCPA. See Streptococcal C5a peptidase Scrambled antigen vaccine, 649 Segmented negative strand RNA virus, 366–367 Selectins, 178 Sendai virus, 366 Serine rich E. histolytica protein (SREHP), 823–826 Seroconversion, 234 Serogroup C disease. See Meningococcal C disease Serogroup C meningococcal disease. See Meningococcal C disease Serotype replacement disease, 469 Serotype-specific immunity, 673 Serratia marcescens, 132 Serum antibody, 533 Serum Anti-Vi IgG response Vi-rEPA conjugate injections, 490, 492 Serum bactericidal activity (SBA), 221, 443 vaccine-induced, 456 Serum hemagglutination inhibition (HAI) test, 533 Severe acute respiratory syndrome (SARS), 82, 119, 914 active immunization, 918 DNA vaccines, 923 immune response to infection, 915 and inactivated SARS-CoV vaccines, 918 inactivated virus vaccine boost and, 923 licensed vaccine for, 923 live attenuated vaccines, 922–923 outbreak of, 914 passive immunization, 917–918 and protective immunity epitopes, identification of, 916 goal of SARS vaccines, 916 structural proteins, role of, 915–916 SARS-coronavirus (SARS-CoV) as causative agent, 914 characteristics of, 915 genome and reservoir of, 914–915 SARS-CoV vaccines animal models, for evaluation of vaccines, 916–917 and development of ADE, 917 preclinical evaluation, of candidate vaccines, 916 subunit vaccines for, 918–920 vaccine strategies for, 919 vectored vaccines for, 920 adenoviruses, 920–921 alphaviruses, 921 paramyxoviruses, 921 poxviruses, 921–922 rhabdovirus, 922 virus, 363, 365, 390 VLP vaccine strategy for, 923 Shiga toxin (Stx), 493–494 Shigella, 13, 26, 30 Shigella dysenteriae, 700 Shigella flexneri, 700, 701, 703, 704, 705, 707, 709, 723 Shigella vaccine, 724 Shigella infection animal models, 703 clinical and epidemiological features, 700 construction of rational live-attenuated shigella strains, 702–703 Escherichia coli carrier vaccine, 705 hybrid vaccines, 704–705 immune mechanisms homotypic vs. heterotypic immunity, 700–701 immunoprotective responses, specific, 701 protective immune responses, 700 immune mechanisms, 700

[Shigella infection] mutants of shigella, enteroinvasive deletion aromatic auxotrophs, 705 purine auxotrophs, 705, 707 pathogenesis inflammatory destruction of the intestinal epithelium, 701–702 invasive phenotype, virulence plasmid of shigella, 701 role of toxin in, 702 shigella fundamentally attenuated with a mutation in virG, 707–708 shigella serotypes, 700 shigella vaccines, polyvalent, 709 shigella vaccine strains lacking invasive phenotype streptomycin-dependent vaccines, 703 T32-Istrati strain, 703 subcellular extracts of virulent shigella, 708–709 vaccine candidates, models for evaluation of volunteer models for vaccine efficacy, 703 Shigella sonnei, 700, 701, 703, 704, 705, 708, 709 Shigella vaccine strains Escherichia coli carrier vaccine, 705 hybrid vaccines, 704–705 lacking invasive phenotype FS Bivalent vaccine, 703–704 streptomycin-dependent vaccines FS Bivalent vaccine, 703–704 T32-Istrati strain, 703 streptomycin-dependent vaccines, 703 vaccines for FS Bivalent vaccine, 703–704 T32-Istrati strain, 703 Signature-tagged mutagenesis (STM), 19, 447 Simian human immunodeficiency virus (SHIV) vaccines, 345 Simian immunodeficiency virus (SIV), 342, 352, 354, 363 Simian virus 5 (SV5), 366 Sindbis, 355 Sindbis replicon-based measles DNA vaccine biodistribution studies, 582 development, 581 efficacy studies, 581–582 immunogenicity studies, 581 integration studies, 582 phase I trial, 582 toxicology studies, 582 Single-nucleotide polymorphisms (SNPs), 18 Single radial immunodiffusion (SRID), 534 Six Sigma, 150 Skin-associated lymphoid tissue (SALT), 415 Skin immunization adjuvants, 417 SLE. See St. Louis encephalitis (SLE); Systemic lupus erythematosus Small Molecular weight Immune Potentiators (SMIPs), 293 Smallpox vaccine, 340, 343, 353 Smallpox vaccines, development of, 838 attenuated strains of live virus, use of, 838 LC16m8, 838–839 MVA, 839 and causative agent, 838, 839 DNA-based subunit vaccines, 846–847 eradication program, 838 live vaccinia virus vaccines, 838 protein-based subunit vaccines, 839 combination gene targets, 839, 843–845 and immune response modifiers (IRMs), 846 individual gene targets, 839–842 monkeypox model, of poxvirus infection, 846 subunit-based vaccines, 839 vectored vaccines, 847

1007

SMFA. See Standard Membrane Feeding Assay (SMFA) SNPs. See Single-nucleotide polymorphisms (SNPs) Societal issues for vaccine industry immunization, access to dual track, 88 intellectual property (IP), 88 technology transfers, 88 tiered pricing, 89 vaccines, perception of, 89 Sortase attachment sites, 15 South Asian Association for Regional Cooperation (SAARC) , 156 SPA-TT, phase I and phase II clinical trials of, 493 SPI 2. See Salmonella pathogenicity island 2 Split influenza vaccine, 327 SRID. See Single radial immunodiffusion (SRID) St. Louis encephalitis (SLE) , 557, 562 Standardization activities, of ECBS, 122 Standard Membrane Feeding Assay (SMFA), 757, 758 Staph enterotoxin, 363 Staphylococcal toxic shock syndrome, 729 Staphylococcal virulence factors, 729–730S 729-730S Staphylococcus aureus, 309 Staphylococcus aureus infection, 8 passive immunotherapies, 732 pathogenesis of clinical manifestations of disease, 728–729 epidemiology of, 729 host defense, 729 staphylococcal virulence factors, 729–730 vaccination, live, 731–732 vaccines for polysaccharide vaccines, 731 protein subunit vaccines, 731 StkP, serine/threonine protein kinase, 485 STM. See Signature-tagged mutagenesis (STM) Stratum corneum (SC), 415 Streptococcal C5a peptidase in emm genotypes, 673 genome plasticity, 673 intranasal immunization with intranasal NALT infection model for, 674 and M protein, 673–674 streptococcal virulence of, 674 vaccines based on, 674–675 Streptococcal M protein coiled-coil rod region, 676 conserved epitope within C-repeat domain of chimeric peptides and, 677 local mucosal response directed to, 680 serotypic determinants and, 677–679 functions of, 677 immune response to conserved region of, 679 as model system, 682 as streptococcal vaccine, 680–681 structure, 676–677 vaccines based on, 674 Streptococcal opsonization, 680 Streptococcal pharyngitis mucosal antibodies, 679 occurrence in child and adult, 679 passive protection against, 679–680 Streptococcus agalaticae, 17 Streptococcus gordonii, 13 Streptococcus gordonii, as vaccine vector, 681 and Lactococcus lactis, 683 safety trials of, 683–684 surface-exposed segment deletion and replacement, 682 translocation of recombinant molecule, 682 Streptococcus mutans, 322

1008

Index

Streptococcus pneumoniae, 13, 17, 76, 105, 235, 473, 474, 482 Pht proteins immunity against, 484 PiuA and PiaA reactive with, 484 Streptococcus pyogenes, 242, 319 Subunit vaccines, for plague, 873–874 Suicidal cassettes, 794–795 Surface proteins in gram-positive bacteria C-terminal hydrophobic domain and charged tail in, 681 synthesis of, 681 Swine fever virus, 364 Swine-transmissible gastroenteritis virus (TGEV), 308 Swine vesicular disease virus, 364 Syntex adjuvant formulation (SAF), 255, 287 Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN), 418 Synthetic peptide–based polymers, preparation of, 317–318 Systemic lupus erythematosus, 242 TAM. See Tumor-associated macrophages (TAM) Tax protein, 243 TB. See Tuberculosis (TB) TBE. See Tick-borne encephalitis (TBE) virus TBE booster vaccination, 237 TBMV. See Transmission-blocking malaria vaccines (TBMV) TB vaccine animal models used for efficacy testing of, 520 BCG, 516, 521 replacement live mycobacterial vaccines, 521–522 types of, 519 booster vaccines, 520, 522 postexposure vaccines, 522 prime vaccines, 520 therapeutic vaccines, 523 T-cell activation, by superantigens, 174 T-cell receptor, 237 T-cell responses in early life challenges to induction of, 230 factors limiting to, 230–231 TCI. See Transcutaneous immunization (TCI) TCR. See T-cell receptor TCR-signaling, 237 Tetanus, 322 toxin, 815 Tetraspanin integral membrane protein, 812 Th cell populations, 174 Th cells. See T helper cells T helper cells characterization, 517 cytokine production by, 518 Th1 cells, 174 Therapeutic vaccines, 294 role as adjunctive therapy against TB, 523 Th2 responses, 175 Thrombospondin-related adhesive protein (TRAP), 398, 752–753 Th1-type adjuvants, 614 Th1-type cell-mediated immunity, 265 Thymopoiesis, 237 Thymus, age-dependent involution of, 238 Tick-borne encephalitis (TBE), 236 virus, 352, 557, 559, 561, 562 T-Independent B-cell responses, 172 TIV. See Trivalent inactivated vaccine (TIV) TLRs. See Toll-like receptors T Lymphocytes, 237–239 memory, 172 TOLAMBA vaccine, 267

Toll-like receptors (TLRs), 251, 285–286, 315 in humans, properties, 167 and recognition of CpG DNA, 263 signaling, 274 Total quality management, 150 Toxoplasma gondii, 266 Toxorhynchites splendens, 562 TQM. See Total quality management Traditional vaccines, advantages over, 273 Transcutaneous immunization (TCI) adjuvants bacterial ADP-ribosylating exotoxins (bAREs), 417–418 CpG motifs, 418 imiquimod, 418 applications for bacterial diseases Bacillus anthracis, 418 chlamydia, 418 cholera, 418 clostridium, 418–419 Haemophilus influenzae, 418 plague, 419 applications for miscellaneous diseases Alzheimer’s disease (AD), 420–421 melanoma, 420 applications for viral diseases herpes simplex, 419 HIV, 419 human papillomavirus, 420 measles virus, 420 pandemic influenza, 420 respiratory syncytial virus, 419 seasonal influenza, 419 virus-like particle (VLPs), 420 approach, 542 clinical product development dry patch, 421 Iomai dry patch vaccines, 422 methods for skin disruption Iomai’s SPS system, 416 physical methodologies, 416 skin structure and immune function, 415–416 Transcutaneous vaccination, 855 Transmission-blocking malaria vaccines (TBMV), 757–758 in clinical development, 760–762 P25 and P28, 761–762 Pfs48/45, 760–761 Pfs230, 761 control of malaria transmission, 758–759 development of, 759–760 targeting ‘‘hot spots’’ of malaria transmission, 759 TRAP. See Thrombospondin-related adhesive protein (TRAP) Traveler’s diarrhea, 308 Traveler’s diarrhea illness LT patch for prevention and reduction of, 425–426 placebo-controlled field trial, 425 Travel vaccines, 236–237 Trehalose, 542 Trichuriasis, 830 Trimeric spike protein vaccine (triSpike), 920 Trivalent inactivated influenza vaccine (TIV), 419 Trivalent inactivated vaccine (TIV), 534 Trypanosoma cruzi, 175, 264 TST. See Tuberculin skin test (TST) Tuberculin skin test (TST), 399 Tuberculosis (TB), 284, 831 antigen presentation in, 517 BCG prime-MVA85A boost trial, 399–400 effector memory T cells and central memory T cells role in, 519

[Tuberculosis (TB)] in humans and animal models, 520 immunologic memory development during, 519 prevalence of, 516 vaccine against. See TB vaccine Tularemia vaccines adaptive immune response, 865 antibodies, 865 bacteriology, 864 killed vaccine, 865 live attenuated vaccine, 865–868 other vaccine strategies, 868 virulence, 864–865 Tumor-associated macrophages (TAM) , 936–937 Tumor cells, vaccines comprised of, 329–330 Ty2 attenuating strategies, 498–499 Ty21a efficacy, 498 enteric-coated formulation, 498 ‘‘liquid suspension’’ formulation of, 498 mutations in, 497 protection correlation of, 498 reactogenicity in adults and children, 497–498 Ty445 and Ty800, 502 541Ty and 543Ty strains, 499 Type 1 cytokine interferon (IFN)-g, 238 Type I diabetes, 242, 246 Typhoid fever incidence rates, 497 target populations, 497 in young children, 489 Typhoid vaccines, 4 immunogenicity, 489 Vi polysaccharide vaccine, 490 Vi-rEPA conjugate vaccine dosage study, 491–492 immunogenicity, 490 infant study, 492 phase I and phase II studies, 490–491 phase III studies, 491 preparation, 490 UCI. See Universal childhood immunization (UCI) UL6 protein, 243 UNAIDS, Joint United Nations Programme on HIV/AIDS, 61, 62, 63 UNICEF. See United nations children’s fund (UNICEF) UNICEF, 67, 73, 79, 82, 83, 92, 93, 94, 97, 98, 101, 103, 104 United nations children’s fund (UNICEF), 156, 162 Universal childhood immunization (UCI), 93 Uvularia grandiflora, 1 Vaccinations, 234, 969, 977–978 and diabetes, 246–247 hepatitis A/B, 236 new delivery systems, 240 against tetanus, diphtheria, poliomyelitis, and, 236 Vaccine adjuvants, 250. See also Adjuvants aluminum compounds, 252–253 AS04, 253–254 classes of, 251 exotoxins, 253 microfluidized oil/water emulsion (MF59), 253 virosomes, 253 allergy. See Allergy vaccines

Index [Vaccine] biomarkers for, 118 booster. See Booster vaccine clinical development. See Vaccine development EBV. See EBV vaccine formulations efficacy, 234, 237 epitope-based, 582 ETEC candidate. See ETEC candidate vaccines GAS. See GAS vaccine HCV. See HCV vaccine, efficacious immunogenicity, 239–240 inactivated ETEC. See Inactivated ETEC vaccines live anthrax. See Live anthrax vaccines live attenuated. See Specific vaccine measles DNA. See Measles DNA vaccine meningococcal C. See Meningococcal C vaccine new generation and early risk assessment, 247 PIV. See PIV vaccines prequalification of, 125–127 recombinant PA–based. See Recombinant PA–based vaccines recommendation for routine use in elderly, 236 regulation, 127–128 in United States, 122 RSV. See RSV vaccines sindbis replicon-based measles DNA. See Sindbis replicon-based measles DNA vaccine TB. See TB vaccine typhoid. See Typhoid vaccines 26-valent M protein–based. See 26-valent M protein–based vaccine Vi-rEPA conjugate. See Vi-rEPA conjugate vaccine Vaccine, administration of mucosal immunization aerosols, vaccines delivered as small-particle, 408–409 duration of protection, 406 immune effector responses, 406 inductive sites for immune responses, 405–406 nasal vaccines, 407 oral vaccines, 406 needle-free devices jet injectors, 409–410 Vaccine, safety adverse effects contamination, 131–132 hypersensitivity reactions, 132 immunologically mediated reactions, 132 inflammatory responses, 132 injection related, 131 replication of live agents, 132 causality assessment criteria for causality, 138 definition of, 134 evolution of clinical pre-licensure studies, 135 passive surveillance systems, 137–138 post-licensure assessment of, 136 preclinical testing, 135 vaccine licensure, 136 institute of medicine safety review committee, 139 perceptions of safety, false, 134 risk management and communication, 139 safety lessons from the history of vaccine development cutter incident, 133 enhancement of measles and respiratory syncytial virus disease, 133

[Vaccine, safety safety lessons from the history of vaccine development] osp A Lyme disease vaccine, 134–135 seed lots and standardization of manufacturing, 133 vaccine-associated paralytic polio, 133 vaccine-derived polio outbreaks, 133 vaccine purity, 132–133 whole-cell pertussis vaccines, 133 safety lessons from the history of vaccine development vaccine, safety rotavirus vaccines, 133–134 Vaccine adjuvants. See also Adjuvant MF59; Antigen delivery systems alternative approaches, 284–286 classification system for, 286 delivery system, 290–293 development, 293 Intercell’s composition, 292 microparticles as, 290 need for new and improved, 284–285 options for, 293 polymeric microparticles for delivery, 290–292 safety, 284 Vaccine Adverse Event Reporting System (VAERS), 26, 54, 118, 133 Vaccine-attributable autoimmune diseases, 242, 245 Vaccine calamities events in the first half of the 20th century disasters following diphtheria immunization Lubeck disaster, 8 Mulkowal disaster, 8 typhoid fever, immunization with a heattreated oral typhoid vaccine, 8 events in the latterhalf of the 20th century cutter incident, 8 swine influenza vaccine and GuillainBarre´ syndrome, 8 Vaccine candidate, clinical evaluation of new clinical trials, phases, 25 comparative genomics pan-genome, 18–19 consent, informed studies involving children and infants, considerations for, 31, 32 facilities, 26 functional genomics global genomic profiling using microarrays, 19–20 protein array technology, 20 proteomics, 20 genomic revolution epitope predictions and immunomics, 17–18 reverse vaccinology, 15–17 probability of success, scoring vaccines for, 21–22 protocol development components of, 29 studies involving children and infants, considerations for, 29 vaccines prepared by recombinant DNA technology, considerations for, 30 vaccines that can be transmitted personto-person, considerations for, 30 recombinant DNA technology, 12–15 regulatory considerations in international trials, 29 regulatory issues investigational new drugs application, components of, 27 regulation of vaccine development, history of, 27

1009

[Vaccine candidate, clinical evaluation of new] selection process, 25 structural prediction and structural genomics, 20 volunteers, selection of studies involving children and infants, considerations for, 31 volunteers, selection of vaccine candidate, clinical evaluation of new, considerations, general 30 Vaccine development, 219 ELISPOT assays, for immunological memory, 223–224 high-throughput assays measuring antibody function, 223 measuring antibody quantity, 222–223 key guidance, 119 measurement of correlates of protection, 220–222 and multivalent trends, 219–220 bivalent Hib-HepB and, 219 Hib vaccines, 219 pentavelent formulation, 219 Pnc vaccines, 220 surrogates, as indicators of protection, 220 vaccine evaluation, 220 meeting demands of, 224 Vaccine economics development costs in a perfect world, 68–69 economic equation, innovative mechanisms to change Advanced Market Commitments (AMC), 74–77 International Finance Facility for Immunization (IFFIm), 73 pull and push mechanism, 73 global vaccine market, 66 pricing equity pricing, 67 price discrimination, 68 tiered pricing, 67–68 suppliers of vaccine, 66–67 vaccine production, 69–72 Vaccine-induced SBA, 456 Vaccine industry, key issues for manufacturing process, 86 marketing process, post, 87–88 research and development process, 83 societal issues immunization, access to, 88–89 perception of vaccines, 89 supply process, 86–87 Vaccine Injury compensation program (VICP), 132, 133 Vaccine-linked chemotherapy, 835 Vaccine manufacturers, 82–83 Vaccine manufacturing bulk manufacture, 145 bacterial fermentation, 145–146 cell culture, 147 cleanliness of operations, 148 inactivation, 147 method of harvest, 147 process, 146 purification, 147 seed preparation, 145 formulation and filling, 147 new trends in disposable materials, 150–151 new trends in, 150 phases and critical issues, 145 raw materials, 145 special circumstances in, 149–150 storage and shipping, 147 testing, 147

1010

Index

Vaccine market closed markets, 79 commercial markets, 80 donors’ markets, 79 growth factors bioterrorism, 82 demography, 81–82 equity, 82 health threats, 82 innovation, 80–81 prevention, 82 increasing value of, 151 projection, 152 2004 vaccine sales by, 151 Vaccine performance, evaluation of post-licensure observational studies methodological approaches, 52–55 reasons for conducting studies of safety and protection, 52 randomized clinical trials of vaccines additional issues for phase III, 43–52 over view of, 42–43 types of evaluations, 41 Vaccine production presentation, 69 reliance on third-party components, 70–71 risks commercial production, 72 delivery and sales, 72 regulatory and licensing issues, 71–72 research and development, 71 scale of operation, 69 supply base location, 71 vaccine batch size, 70 vaccine production characteristics, 71 Vaccines. See also Plant-derived vaccines; Vaccine adjuvants adjuvants role in development, 284–285 Ads biology and vector development for, 352–353 anti-DEC-205 hybrid antibodies as, 331–333 comprised of dying tumor cells and stimulus for NKT lymphocytes, 330 irradiated tumor cells transduced with cytokines, 329–330 dendritic cells based, 327–335 composed, 329 exposed to antigens ex vivo, 329 receptors for targeting protein, 334–335 targeting of tumor cell, 329–330 against Entamoeba histolytica, 820–826 oral, 825–826 parenteral, 824–825 for hookworm infection. See Human hookworm vaccine (HHV) influenza, 288–289 against leishmaniasis. See Leishmaniasis lipopeptide-based, 315–323 against plague, 873–875 production and delivery in plants, 306 proteins targeting to maturing DCs in vivo, 330–333 vectors from RNA virus, 361–365 Vaccine Safety Data Link (VSD), 26 Vaccines and Related Biological Products Advisory Committee (VRBPAC), 118 Vaccines and vaccination, historical perspective of immunizing agents, parenteral and oral attenuated bacteria, 3 live virus vaccines, 4 parenteral inactivated whole-cell bacterial vaccines, 4 subunit and extract vaccines, 4–5

[Vaccines and vaccination, historical perspective of] safety and efficacy of vaccine candidates, clinical evaluation of, 6 vaccination, origin of, 2–3 vaccine calamities events in the first half of the 20th century, 8 events in the latterhalf of the 20th century, 8 vaccine efficacy, large-scale field trials to assess, 6–7 Vaccine trials in developing countries trial design and implementation, issues related to community participation, 37 cultural differences, 38 disease burden data, 36–37 sustainability of study-related activities/ standard of care, 38 trial end points, 37 women’s participation in research, 37–38 trial oversight, issues related to data safety monitoring boards (DSMBs), 39 ethical review committees and institutional review boards, 38 trial site preparation, issues related to, vaccine trials in developing countries informatics, 36 infrastructure, 34–36 trial site preparation, issues related to environment, 36 Vaccinia virus. See also Poxviruses gene expression, 341 HBsAg recombinant, 342 influenza HA recombinant, 342 as live vectors, 340–345 measles recombinants, 343 rabies glycoprotein recombinants, 342 recombinants. See Recombinant vaccinia virus RSV recombinants, 342 vectoring M protein with, 680–681 Vaccinology, 191 reverse, MenB, pioneering work with, 16–17 VAERS. See Vaccine Adverse Event Reporting System (VAERS) 26-Valent M protein–based vaccine, 684 composition, 685 protective efficacy of, 685 safety and immunogenicity of, 686 Validation protocols, 149 Varicella, 353 Varicella-zoster virus, 234, 661 Variolation, 1–2 Variola virus (VARV), 340, 838 VARV. See Variola virus (VARV) Vascular endothelial growth factor (VEGF) , 934 VEE. See Venezuelan equine encephalitis (VEE) VEGF. See Vascular endothelial growth factor (VEGF) Venezuelan equine encephalitis (VEE), 638, 880, 898 virus, 355, 589 Vertebrate host immune system, 191–192 Vesicular stomatitis virus (VSV), 352, 365–366 Vibrio cholerae, 13, 20, 30, 45, 46, 51, 308–309, 406, 418. See also Cholera Disease Vi conjugate vaccine, 489

VICP. See Vaccine injury compensation program (VICP) Vi polysaccharide vaccine efficacy trials, 490 Vi-rEPA conjugate vaccine dosage-immunogenicity study of, 491–492 immunogenicity, 490 infant study, 492 phase I and phase II studies, 490–491 phase III studies, 491 preparation, 490 Virosomal vaccines, 290 Virosomes, 253, 286, 290, 543 Virus-like particles (VLPs), 284, 286, 288, 292–293 vaccine carriers Hepatitis B virus VLPs, 301–302 influenza M2 protein, 302 malaria antigens, 302 Rotavirus VLP-based vaccines, 301 vaccine against Alzheimer’s disease, 302–303 vaccine for nicotine addiction, 303 VLP-Based Vaccines Hepatitis B virus vaccines, 299–300 human papillomavirus vaccines, 300 Norwalk virus and noroviruses, vaccines for, 300–301 Rotavirus VLP-based vaccines, 301 VLP-Based Vaccines, Virus-like replicon particles (VRP), 638, 898 Visceral leishmaniasis (VL), 790–792, 796–797, 799 VLPs. See Virus-like particles (VLPs) Volunteers selection for clinical trials of new vaccine candidate considerations, general, 30 studies involving children and infants, considerations for, 31 VRBPAC. See Vaccines and Related Biological Products Advisory Committee VRP. See Virus-like replicon particles (VRP) VSD. See Vaccine Safety Data Link (VSD) VZV. See Varicella-zoster virus Walter Reed Army Institute of Research (WRAIR), 596 Warts, 954 Water for injection, 149 West Nile virus (WNV), 389–390 chimerivax vaccines ChimeriVax-WN, 566 structure and replication of, flavivirus, 557–559 Wheezing, and CAIV, 539 WHO. See World Health Organization WHO documents for specific vaccines, 122 Whole-virus vaccines, 14 Window of vulnerability definition, 579 immunizing young infants during, 579–580 WN virus. See West Nile virus(WNV) Working cell bank, 147 World Health Organization (WHO), 37, 41, 67, 74, 75, 79, 80, 82, 86, 91, 92, 93, 94, 95, 96 97, 98, 103, 104, 119, 121 collaborating centers, 224 NRA strengthening activity, 123–125, 129 on quality assurance of vaccines, 121–122

Index WRAIR. See Walter Reed Army Institute of Research (WRAIR) X3927, X4073, and X8110 (live oral typhoid vaccine), 502 X-linked lymphoproliferative (XLP) disease incidence of, 646

Yeast-derived hepatitis B vaccine, 306 Yellow fever (YF), 236, 353 vaccines, 96, 98, 132 virus, 560 Yersinia enterocolitica, 871, 873–874

1011

Yersinia pestis, 308–309, 419 lifestyle, 872–873 and plague, 871 virulence factors and protective antigens, 871–872 Yersinia pseudotuberculosis, 871–874 YF. See Yellow Fever (YF) virus

Sheridan_8.5x11

Infectious Disease

The Fourth Edition delivers critical updates to both clinicians and researchers on: • r ecently licensed vaccines, including a quadrivalent (groups A, C, W135, and Y) meningococcal conjugate vaccine, two oral rotavirus vaccines, and two human papillomavirus (HPV) vaccines based on virus-like particle technology •m  odifications to existing vaccines, such as drugs that have been adapted from use in infants, for use in adolescents and adults •n  ew technologies that allow high-throughput sequencing and bioinformatic analysis of genome sequence data •u  pdates on the GAVI Alliance and its partners, including the World Health Organization (WHO), UNICEF, the World Bank, and the Bill and Melinda Gates Foundation •“  vaccine safety” in industrialized countries versus the developing world, and the requirements of regulatory agencies such as WHO and the FDA about the editor-in-chief... MYRON M. LEVINE is Director, Center for Vaccine Development; Grollman Distinguished Professor of Medicine; Head, the Division of Geographic Medicine; and Professor of Pediatrics, Microbiology and Immunology, and Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA. He received his M.D. from the Medical College of Virginia, Richmond, Virginia, USA and D.T.P.H. from the London School of Hygiene and Tropical Medicine, London, UK. Dr. Levine is on the editorial boards of Vaccine, Human Vaccines, and Public Health Reviews. He is a consultant to many organizations, including WHO, NIH, the Vaccine Research Center, Institute of Medicine, and the U.S. Department of Defense, and sits on the scientific advisory boards of multiple vaccine biotechnology companies. Dr. Levine is a member of numerous societies, including the Institute of Medicine of the National Academy of Sciences, the American Society of Clinical Investigation, and the Academy of Microbiology. He is past President of the American Epidemiological Society and of the American Society of Tropical Medicine and Hygiene. about the editors... GORDON DOUGAN is Head, Microbial Pathogenesis Group, and Board of Management Representative of Pathogens, The Wellcome Trust Sanger Institute, Cambridge, UK.

New Generation Vaccines

The latest edition of New Generation Vaccines reflects important changes that have taken place in the field since the prior edition. Comprised of 89 chapters, this all-encompassing text is written by 250 of the world’s top experts in vaccinology, infectious disease, immunology, virology, microbiology, tropical medicine, biologicals, public health, and pediatrics.

Fourth Edition

about the book…

New Generation Vaccines Fourth Edition

MICHAEL F. GOOD is Director, Queensland Institute of Medical Research, Queensland, Australia. MARGARET A. LIU, ProTherImmune, Lafayette, California, USA, and Karolinska Institutet, Stockholm, Sweden. GARY J. NABEL is Director and Chief, Laboratory of Virology and Vector Core Laboratory, Vaccine Research Center, National Institutes of Health, Bethesda, Maryland, USA. JAMES P. NATARO is Professor of Pediatrics; Chief, Section of Molecular Diagnostics, Center for Vaccine Development; and Head, Pediatric Infectious Diseases and Tropical Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, USA. RINO RAPPUOLI is Global Head, Vaccines Research, Novartis Vaccines and Diagnostics, Siena, Italy. Printed in the United States of America Cover illustrations by Jeff Priester.

H6073

Levine •

Dougan Good Liu Nabel Nataro Rappuoli

Editor-in-Chief

Myron M. Levine Editors

Gordon Dougan Michael F. Good Margaret A. Liu

Levine_978-1420060737.indd 1

Gary J. Nabel James P. Nataro Rino Rappuoli

11/10/09 10:08:00 AM

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