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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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 (Saccha